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Sample records for naked poliovirus replicons

  1. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons.

    PubMed

    Jackson, Cheryl A; Messinger, Jeff; Palmer, Matthew T; Peduzzi, Jean D; Morrow, Casey D

    2003-09-15

    The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or

  2. Construction and characterization of poliovirus subgenomic replicons

    SciTech Connect

    Kaplan, G.; Racaniello, V.R. )

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of ({sup 35}S-labeled) viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A{sup pro}, 2C, and 3D{sup pol} and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs.

  3. Conserved aspartic acid 233 and alanine 231 are not required for poliovirus polymerase function in replicons

    PubMed Central

    Freistadt, Marion S; Eberle, Karen E

    2007-01-01

    Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function. PMID:17352827

  4. Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

    PubMed Central

    Porter, D C; Ansardi, D C; Morrow, C D

    1995-01-01

    Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated

  5. Naked sunspots

    NASA Technical Reports Server (NTRS)

    Liggett, M.; Zirin, H.

    1983-01-01

    Naked sunspots are spots seen in H-alpha to be devoid of associated plage. In magnetograms and K-line little if any opposite polarity field is found, and in soft X ray images a blank appears in the region of the spot. In almost all cases studied in which naked spots resulted the spot groups had emerged in unipolar regions of the same polarity as the naked spot. At least half of the naked spots are associated with coronal holes. The naked spots are long-lived and show rotation rates close to the Newton-Nunn curve. Most of the naked spots had bright rims in H-alpha, and the one spot observed to disappear left no trace in the background magnetic field. These spots may be a means by which separation of p from f magnetic polarity occurs.

  6. Naked sunspots

    NASA Technical Reports Server (NTRS)

    Liggett, M.; Zirin, H.

    1983-01-01

    Naked sunspots are spots seen in H-alpha to be devoid of associated plage. In magnetograms and K-line little if any opposite polarity field is found, and in soft X ray images a blank appears in the region of the spot. In almost all cases studied in which naked spots resulted the spot groups had emerged in unipolar regions of the same polarity as the naked spot. At least half of the naked spots are associated with coronal holes. The naked spots are long-lived and show rotation rates close to the Newton-Nunn curve. Most of the naked spots had bright rims in H-alpha, and the one spot observed to disappear left no trace in the background magnetic field. These spots may be a means by which separation of p from f magnetic polarity occurs.

  7. Replicon RNA Viral Vectors as Vaccines

    PubMed Central

    Lundstrom, Kenneth

    2016-01-01

    Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions. PMID:27827980

  8. 'Naked' radiopharmaceuticals

    SciTech Connect

    Wallner, Paul E. . E-mail: pwallner@rtsx.com

    2006-10-01

    The term 'naked' radiopharmaceuticals, more appropriately, 'unbound' radiopharmaceuticals, refers to any radioisotope used for clinical research or clinical purposes that is not attached to a chemical or biological carrier, and that localizes in various tissues because of a physiologic or chemical propensity/affinity, or secondary to focal anatomic placement. Although they remain useful in selected clinical circumstances, the available agents (except for Iodine-131) have been relegated to an unfortunate and somewhat secondary role. The agents remain useful and worthy of consideration for new clinical investigation and clinical use.

  9. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    PubMed

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax.

  10. Chromosomal replicons of higher plants

    SciTech Connect

    Van't Hof, J.

    1987-03-16

    This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs.

  11. Replicon system for Lassa virus.

    PubMed

    Hass, Meike; Gölnitz, Uta; Müller, Stefanie; Becker-Ziaja, Beate; Günther, Stephan

    2004-12-01

    Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

  12. Timelike naked singularity

    SciTech Connect

    Goswami, Rituparno; Joshi, Pankaj S.; Vaz, Cenalo; Witten, Louis

    2004-10-15

    We construct a class of spherically symmetric collapse models in which a naked singularity may develop as the end state of collapse. The matter distribution considered has negative radial and tangential pressures, but the weak energy condition is obeyed throughout. The singularity forms at the center of the collapsing cloud and continues to be visible for a finite time. The duration of visibility depends on the nature of energy distribution. Hence the causal structure of the resulting singularity depends on the nature of the mass function chosen for the cloud. We present a general model in which the naked singularity formed is timelike, neither pointlike nor null. Our work represents a step toward clarifying the necessary conditions for the validity of the Cosmic Censorship Conjecture.

  13. Detection of Imported Wild Polioviruses and of Vaccine-Derived Polioviruses by Environmental Surveillance in Egypt

    PubMed Central

    El Bassioni, Laila; El Maamoon Nasr, Eman M.; Paananen, Anja; Kaijalainen, Svetlana; Asghar, Humayun; de Gourville, Esther; Roivainen, Merja

    2012-01-01

    Systematic environmental surveillance for poliovirus circulation has been conducted in Egypt since 2000. The surveillance has revealed three independent importations of wild-type poliovirus. In addition, several vaccine-derived polioviruses have been detected in various locations in Egypt. In addition to acute flaccid paralysis (AFP) surveillance, environmental surveillance can be used to monitor the wild poliovirus and vaccine-derived poliovirus circulation in populations in support of polio eradication initiatives. PMID:22582070

  14. Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

    PubMed

    Sosnovtseva, A O; Lipatova, A V; Grinenko, N F; Baklaushev, V P; Chumakov, P M; Chekhonin, V P

    2016-10-01

    A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

  15. Antiviral activity of lactoferrin towards naked viruses.

    PubMed

    Seganti, Lucilla; Di Biase, Assunta Maria; Marchetti, Magda; Pietrantoni, Agostina; Tinari, Antonella; Superti, Fabiana

    2004-06-01

    It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

  16. Naked Black Hole Firewalls.

    PubMed

    Chen, Pisin; Ong, Yen Chin; Page, Don N; Sasaki, Misao; Yeom, Dong-Han

    2016-04-22

    In the firewall proposal, it is assumed that the firewall lies near the event horizon and should not be observable except by infalling observers, who are presumably terminated at the firewall. However, if the firewall is located near where the horizon would have been, based on the spacetime evolution up to that time, later quantum fluctuations of the Hawking emission rate can cause the "teleological" event horizon to have migrated to the inside of the firewall location, rendering the firewall naked. In principle, the firewall can be arbitrarily far outside the horizon. This casts doubt about the notion that firewalls are the "most conservative" solution to the information loss paradox.

  17. Naked Black Hole Firewalls

    NASA Astrophysics Data System (ADS)

    Chen, Pisin; Ong, Yen Chin; Page, Don N.; Sasaki, Misao; Yeom, Dong-han

    2016-04-01

    In the firewall proposal, it is assumed that the firewall lies near the event horizon and should not be observable except by infalling observers, who are presumably terminated at the firewall. However, if the firewall is located near where the horizon would have been, based on the spacetime evolution up to that time, later quantum fluctuations of the Hawking emission rate can cause the "teleological" event horizon to have migrated to the inside of the firewall location, rendering the firewall naked. In principle, the firewall can be arbitrarily far outside the horizon. This casts doubt about the notion that firewalls are the "most conservative" solution to the information loss paradox.

  18. Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

    PubMed

    Yu, Yun-Zhou; Guo, Jin-Peng; An, Huai-Jie; Zhang, Shu-Ming; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-05-07

    Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

  19. Photodynamic Effect of some Phthalocyanines on Enveloped and Naked Viruses.

    PubMed

    Nikolaeva-Glomb, L; Mukova, L; Nikolova, N; Kussovski, V; Doumanova, L; Mantareva, V; Angelov, I; Wöhrle, D; Galabov, A S

    2017-01-01

    Activity of three photosensitizing phthalocyanine derivatives was tested for photodynamic inactivation towards two coated and two non-enveloped viruses - bovine viral diarrhea virus (BVDV), influenza virus A(H3N2), poliovirus type 1 (PV-1) and human adenovirus type 5 (HAdV5). In the case of coated viruses, a combination of virucidal and irradiation effects was registered by octa-methylpyridyloxy-substituted Ga phthalocyanine (Ga8) toward BVDV, whereas tetra-methylpyridyloxy-substituted Ga phthalocyanine (Ga4) revealed a marked photoinactivation only. No such effect was observed towards influenza A virus. In contrast, the photoinactivating potential of Ga4 and Ga8 marked very high values on naked viruses, especially on HAdV5, at which both the virucidal as well as the irradiation effects became combined.

  20. The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

    PubMed Central

    Téoulé, François; Brisac, Cynthia; Pelletier, Isabelle; Vidalain, Pierre-Olivier; Jégouic, Sophie; Mirabelli, Carmen; Bessaud, Maël; Combelas, Nicolas; Autret, Arnaud; Tangy, Frédéric; Delpeyroux, Francis

    2013-01-01

    We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. PMID:23926333

  1. Standardized Methods for Detection of Poliovirus Antibodies.

    PubMed

    Weldon, William C; Oberste, M Steven; Pallansch, Mark A

    2016-01-01

    Testing for neutralizing antibodies against polioviruses has been an established gold standard for assessing individual protection from disease, population immunity, vaccine efficacy studies, and other vaccine clinical trials. Detecting poliovirus specific IgM and IgA in sera and mucosal specimens has been proposed for evaluating the status of population mucosal immunity. More recently, there has been a renewed interest in using dried blood spot cards as a medium for sample collection to enhance surveillance of poliovirus immunity. Here, we describe the modified poliovirus microneutralization assay, poliovirus capture IgM and IgA ELISA assays, and dried blood spot polio serology procedures for the detection of antibodies against poliovirus serotypes 1, 2, and 3.

  2. Naked singularities as particle accelerators

    SciTech Connect

    Patil, Mandar; Joshi, Pankaj S.

    2010-11-15

    We investigate here the particle acceleration by naked singularities to arbitrarily high center of mass energies. Recently it has been suggested that black holes could be used as particle accelerators to probe the Planck scale physics. We show that the naked singularities serve the same purpose and probably would do better than their black hole counterparts. We focus on the scenario of a self-similar gravitational collapse starting from a regular initial data, leading to the formation of a globally naked singularity. It is seen that when particles moving along timelike geodesics interact and collide near the Cauchy horizon, the energy of collision in the center of mass frame will be arbitrarily high, thus offering a window to Planck scale physics.

  3. MAGNETIC TOPOLOGY OF A NAKED SUNSPOT: IS IT REALLY NAKED?

    SciTech Connect

    Sainz Dalda, A.; Vargas Dominguez, S.; Tarbell, T. D.

    2012-02-10

    The high spatial, temporal, and spectral resolution achieved by Hinode instruments gives much better understanding of the behavior of some elusive solar features, such as pores and naked sunspots. Their fast evolution and, in some cases, their small sizes have made their study difficult. The moving magnetic features (MMFs) have been studied during the last 40 years. They have been always associated with sunspots, especially with the penumbra. However, a recent observation of a naked sunspot (one with no penumbra) has shown MMF activity. The authors of this reported observation expressed their reservations about the explanation given to the bipolar MMF activity as an extension of the penumbral filaments into the moat. How can this type of MMF exist when a penumbra does not? In this Letter, we study the full magnetic and (horizontal) velocity topology of the same naked sunspot, showing how the existence of a magnetic field topology similar to that observed in sunspots can explain these MMFs, even when the intensity map of the naked sunspot does not show a penumbra.

  4. Inactivation of poliovirus by formaldehyde

    PubMed Central

    Gard, Sven

    1957-01-01

    Since formaldehyde, either alone or in combination with other inactivating agents, is at present used in the production of all so-called “killed” poliovirus vaccines, a thorough knowledge of the kinetics of the reaction between the chemical agent and the virus, and of the mechanisms involved, is of great practical importance. In this paper the problem is discussed against the background of present knowledge of the structure of the virus and the chemical nature of the action of formaldehyde. PMID:13511143

  5. Functional domains of the poliovirus receptor

    SciTech Connect

    Koike, Satoshi; Ise, Iku; Nomoto, Akio )

    1991-05-15

    A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.

  6. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  7. In vitro replication of poliovirus

    SciTech Connect

    Lubinski, J.M.

    1986-01-01

    Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis of the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.

  8. Development of Plaque Assay Systems for Poliovirus.

    DTIC Science & Technology

    1982-04-01

    from as little as 25 mg/mL (1/10th of that used in the actual plaque assay) to 1000 mg/mL, and to inhibit poliovirus cytopathic effect In...1067 DEVELOPMENT OF PLAQUE ASSAY SYSTEMS FOR POLIOVIRUS (U) by R.E. Fulton and K. Munroe Abstract During the summer months of 1978, Ms. Krista Munroe...quantitation of infectious poliovirus type 1. .Two different plaque assay techniques were developed and compared. The results of this work are presented

  9. Current status of poliovirus infections.

    PubMed

    Melnick, J L

    1996-07-01

    Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus.

  10. Current status of poliovirus infections.

    PubMed Central

    Melnick, J L

    1996-01-01

    Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus. PMID:8809461

  11. Shadow of a naked singularity

    NASA Astrophysics Data System (ADS)

    Ortiz, Néstor; Sarbach, Olivier; Zannias, Thomas

    2015-08-01

    We analyze the redshift suffered by photons originating from an external source, traversing a collapsing dust cloud, and finally being received by an asymptotic observer. In addition, we study the shadow that the collapsing cloud casts on the sky of the asymptotic observer. We find that the resulting redshift and properties of the shadow depend crucially on whether the final outcome of the complete gravitational collapse is a black hole or a naked singularity. In the black hole case, the shadow is due to the high redshift acquired by the photons as they approach the event horizon, implying that their energy is gradually redshifted toward zero within a few crossing times associated with the event horizon radius. In contrast to this, a naked singularity not only absorbs photons originating from the source, but it also emits infinitely redshifted photons with and without angular momenta. This emission introduces an abrupt cutoff in the frequency shift of the photons detected in directions close to the radial one, and it is responsible for the shadow masking the source in the naked singularity case. Furthermore, even though the shadow forms and begins to grow immediately after the observer crosses the Cauchy horizon, it takes many more crossing times than in the black hole case for the source to be occulted from the observer's eyes. We discuss possible implications of our results for testing the weak cosmic censorship hypothesis. Even though at late times the image of the source perceived by the observer looks the same in both cases, the dynamical formation of the shadow and the redshift images has distinct features and time scales in the black hole versus the naked singularity case. For stellar collapse, these time scales seem to be too short to be resolved with existing technology. However, our results may be relevant for the collapse of seeds leading to supermassive black holes.

  12. Will containment of wild poliovirus in laboratories and inactivated poliovirus vaccine production sites be effective for global certification?

    PubMed Central

    Dowdle, Walter R.; Wolff, Christopher; Sanders, Raymond; Lambert, Scott; Best, Maureen

    2004-01-01

    The absolute laboratory containment of any virus cannot be guaranteed, but a wealth of experience indicates that effective containment of wild poliovirus materials for global certification is technically and operationally feasible. Effective containment is based on the principles of minimal wild poliovirus infectious and potentially infectious materials in laboratories; minimal risks of operations in laboratories and inactivated poliovirus vaccine production facilities; minimal susceptibility of workers to wild poliovirus infection and shedding; and minimal susceptibility of populations to wild poliovirus spread. Each principle alone is imperfect, but collectively they greatly minimize the risks of transmitting wild poliovirus from the laboratory to the community. PMID:15106302

  13. The potential benefits of a new poliovirus vaccine for long-term poliovirus risk management.

    PubMed

    Duintjer Tebbens, Radboud J; Thompson, Kimberly M

    2016-12-01

    To estimate the incremental net benefits (INBs) of a hypothetical ideal vaccine with all of the advantages and no disadvantages of existing oral and inactivated poliovirus vaccines compared with current vaccines available for future outbreak response. INB estimates based on expected costs and polio cases from an existing global model of long-term poliovirus risk management. Excluding the development costs, an ideal poliovirus vaccine could offer expected INBs of US$1.6 billion. The ideal vaccine yields small benefits in most realizations of long-term risks, but great benefits in low-probability-high-consequence realizations. New poliovirus vaccines may offer valuable insurance against long-term poliovirus risks and new vaccine development efforts should continue as the world gathers more evidence about polio endgame risks.

  14. Naked singularity resolution in cylindrical collapse

    SciTech Connect

    Kurita, Yasunari; Nakao, Ken-ichi

    2006-03-15

    In this paper, we study the gravitational collapse of null dust in cylindrically symmetric spacetime. The naked singularity necessarily forms at the symmetry axis. We consider the situation in which null dust is emitted again from the naked singularity formed by the collapsed null dust and investigate the backreaction by this emission for the naked singularity. We show a very peculiar but physically important case in which the same amount of null dust as that of the collapsed one is emitted from the naked singularity as soon as the ingoing null dust hits the symmetry axis and forms the naked singularity. In this case, although this naked singularity satisfies the strong curvature condition by Krolak (limiting focusing condition), geodesics which hit the singularity can be extended uniquely across the singularity. Therefore, we may say that the collapsing null dust passes through the singularity formed by itself and then leaves for infinity. Finally, the singularity completely disappears and the flat spacetime remains.

  15. Quantum dress for a naked singularity

    NASA Astrophysics Data System (ADS)

    Casals, Marc; Fabbri, Alessandro; Martínez, Cristián; Zanelli, Jorge

    2016-09-01

    We investigate semiclassical backreaction on a conical naked singularity space-time with a negative cosmological constant in (2 + 1)-dimensions. In particular, we calculate the renormalized quantum stress-energy tensor for a conformally coupled scalar field on such naked singularity space-time. We then obtain the backreacted metric via the semiclassical Einstein equations. We show that, in the regime where the semiclassical approximation can be trusted, backreaction dresses the naked singularity with an event horizon, thus enforcing (weak) cosmic censorship.

  16. Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans.

    PubMed Central

    Ansardi, D C; Porter, D C; Morrow, C D

    1993-01-01

    Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of

  17. Naked singularities and quantum gravity

    SciTech Connect

    Harada, Tomohiro; Iguchi, Hideo; Nakao, Ken-ichi; Singh, T. P.; Tanaka, Takahiro; Vaz, Cenalo

    2001-08-15

    There are known models of spherical gravitational collapse in which the collapse ends in a naked shell-focusing singularity for some initial data. If a massless scalar field is quantized on the classical background provided by such a star, it is found that the outgoing quantum flux of the scalar field diverges in the approach to the Cauchy horizon. We argue that the semiclassical approximation (i.e., quantum field theory on a classical curved background) used in these analyses ceases to be valid about one Planck time before the epoch of naked singularity formation, because by then the curvature in the central region of the star reaches the Planck scale. It is shown that during the epoch in which the semiclassical approximation is valid, the total emitted energy is about one Planck unit, and is not divergent. We also argue that back reaction in this model does not become important so long as gravity can be treated classically. It follows that the further evolution of the star will be determined by quantum gravitational effects, and without invoking quantum gravity it is not possible to say whether the star radiates away on a short time scale or settles down into a black hole state.

  18. Poliovirus Adsorption by 34 Minerals and Soils

    PubMed Central

    Moore, Rebecca S.; Taylor, Dene H.; Sturman, Lawrence S.; Reddy, Michael M.; Fuhs, G. Wolfgang

    1981-01-01

    The adsorption of radiolabeled infectious poliovirus type 2 by 34 well-defined soils and mineral substrates was analyzed in a synthetic freshwater medium containing 1 mM CaCl2 and 1.25 mM NaHCO3 at pH 7. In a model system, adsorption of poliovirus by Ottawa sand was rapid and reached equilibrium within 1 h at 4°C. Near saturation, the adsorption could be described by the Langmuir equation; the apparent surface saturation was 2.5 × 106 plaque-forming units of poliovirus per mg of Ottawa sand. At low surface coverage, adsorption was described by the Freundlich equation. The soils and minerals used ranged from acidic to basic and from high in organic content to organic free. The available negative surface charge on each substrate was measured by the adsorption of a cationic polyelectrolyte, polydiallyldimethylammonium chloride. Most of the substrates adsorbed more than 95% of the virus. In general, soils, in comparison with minerals, were weak adsorbents. Among the soils, muck and Genesee silt loam were the poorest adsorbents; among the minerals, montmorillonite, glauconite, and bituminous shale were the least effective. The most effective adsorbents were magnetite sand and hematite, which are predominantly oxides of iron. Correlation coefficients for substrate properties and virus adsorption revealed that the elemental composition of the adsorbents had little effect on poliovirus uptake. Substrate surface area and pH, by themselves, were not significantly correlated with poliovirus uptake. A strong negative correlation was found between poliovirus adsorption and both the contents of organic matter and the available negative surface charge on the substrates as determined by their capacities for adsorbing the cationic polyelectrolyte, polydiallyldimethylammonium chloride. PMID:6274259

  19. Synchronization of replicons in Ehrlich ascites cells

    SciTech Connect

    Gekeler, V.; Probst, H. )

    1988-03-01

    Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G{sub 1} cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 {mu}M) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G{sub 1} cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles high synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.

  20. Poliovirus Laboratory Based Surveillance: An Overview.

    PubMed

    Zaidi, Syed Sohail Zahoor; Asghar, Humayun; Sharif, Salmaan; Alam, Muhammad Masroor

    2016-01-01

    World Health Assembly (WHA) in 1988 encouraged the member states to launch Global Polio Eradication Initiative (GPEI) (resolution WHA41.28) against "the Crippler" called poliovirus, through strong routine immunization program and intensified surveillance systems. Since its launch, global incidence of poliomyelitis has been reduced by more than 99 % and the disease squeezed to only three endemic countries (Afghanistan, Pakistan, and Nigeria) out of 125. Today, poliomyelitis is on the verge of eradication, and their etiological agents, the three poliovirus serotypes, are on the brink of extinction from the natural environment. The last case of poliomyelitis due to wild type 2 strain occurred in 1999 in Uttar Pradesh, India whereas the last paralytic case due to wild poliovirus type 3 (WPV3) was seen in November, 2012 in Yobe, Nigeria. Despite this progress, undetected circulation cannot fully rule out the eradication as most of the poliovirus infections are entirely subclinical; hence sophisticated environmental surveillance is needed to ensure the complete eradication of virus. Moreover, the vaccine virus in under-immunized communities can sometimes revert and attain wild type characteristics posing a big challenge to the program.

  1. The poliovirus 135S particle is infectious.

    PubMed Central

    Curry, S; Chow, M; Hogle, J M

    1996-01-01

    The molecular mechanism of cell entry by unenveloped viruses is poorly understood. The picornaviruses poliovirus, human rhinovirus, and coxsackievirus convert to an altered form (the 135S or A particle) upon interaction with receptors on susceptible cells at 37 degrees C. The 135S particle is thought to be a necessary intermediate because it accumulates inside susceptible cells soon after infection and drugs which inhibit conversion of the virus to this form also prevent infection. However, since a variable fraction of the altered 135S particles is reported to elute unproductively from the surface of susceptible cells, their precise role remains unclear. We have found that poliovirus 135S particles can infect Chinese hamster ovary (CHO) and murine L cells, neither of which are susceptible to infection by native poliovirus. The infectivity of the particles in tissue culture appears to be between 10(3) to 10(5) times less than that of poliovirus on HeLa cells. The 135S particle infectivity was not sensitive to RNase but was greatly reduced by proteolytic treatment. Proteolysis specifically removed the newly exposed N terminus of VP1, a feature which has previously been shown to mediate interactions of the particle with lipid membranes. These results demonstrate that although the infectivity of the 135S particle appears to be receptor independent, it nonetheless requires some property associated with the protein coat. In particular, the N terminus of VP1 plays an important role in the infection process. Our findings are consistent with the hypothesis that the 135S particle is an intermediate in the normal cell entry pathway of poliovirus infection. PMID:8794359

  2. Has Wild Poliovirus Been Eliminated from Nigeria?

    PubMed

    Famulare, Michael

    2015-01-01

    Wild poliovirus type 3 (WPV3) has not been seen anywhere since the last case of WPV3-associated paralysis in Nigeria in November 2012. At the time of writing, the most recent case of wild poliovirus type 1 (WPV1) in Nigeria occurred in July 2014, and WPV1 has not been seen in Africa since a case in Somalia in August 2014. No cases associated with circulating vaccine-derived type 2 poliovirus (cVDPV2) have been detected in Nigeria since November 2014. Has WPV1 been eliminated from Africa? Has WPV3 been eradicated globally? Has Nigeria interrupted cVDPV2 transmission? These questions are difficult because polio surveillance is based on paralysis and paralysis only occurs in a small fraction of infections. This report provides estimates for the probabilities of poliovirus elimination in Nigeria given available data as of March 31, 2015. It is based on a model of disease transmission that is built from historical polio incidence rates and is designed to represent the uncertainties in transmission dynamics and poliovirus detection that are fundamental to interpreting long time periods without cases. The model estimates that, as of March 31, 2015, the probability of WPV1 elimination in Nigeria is 84%, and that if WPV1 has not been eliminated, a new case will be detected with 99% probability by the end of 2015. The probability of WPV3 elimination (and thus global eradication) is > 99%. However, it is unlikely that the ongoing transmission of cVDPV2 has been interrupted; the probability of cVDPV2 elimination rises to 83% if no new cases are detected by April 2016.

  3. RNA Replicons - A New Approach for Influenza Virus Immunoprophylaxis

    PubMed Central

    Zimmer, Gert

    2010-01-01

    RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed. PMID:21994644

  4. RNA replicons - a new approach for influenza virus immunoprophylaxis.

    PubMed

    Zimmer, Gert

    2010-02-01

    RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed.

  5. Decay of Sabin inactivated poliovirus vaccine (IPV)-boosted poliovirus antibodies

    PubMed Central

    Resik, Sonia; Tejeda, Alina; Fonseca, Magile; Sein, Carolyn; Hung, Lai Heng; Martinez, Yenisleidys; Diaz, Manuel; Okayasu, Hiromasa; Sutter, Roland W.

    2015-01-01

    Introduction We conducted a follow-on study to a phase I randomized, controlled trial conducted in Cuba, 2012, to assess the persistence of poliovirus antibodies at 21–22 months following booster dose of Sabin-IPV compared to Salk-IPV in adults who had received multiple doses of oral poliovirus vaccine (OPV) during childhood. Methods In 2012, 60 healthy adult males aged 19–23 were randomized to receive one booster dose, of either Sabin-inactivated poliovirus vaccine (Sabin-IPV), adjuvanted Sabin-IPV (aSabin-IPV), or conventional Salk-IPV. In the original study, blood was collected at days 0 (before) and 28 (after vaccination), respectively. In this study, an additional blood sample was collected 21–22 months after vaccination, and tested for neutralizing antibodies to Sabin poliovirus types 1, 2 and 3. Results We collected sera from 59/60 (98.3%) subjects; 59/59 (100%) remained seropositive to all poliovirus types, 21–22 months after vaccination. The decay curves were very similar among the study groups. Between day 28 and 21–22 months, there was a reduction of ⩾87.4% in median antibody levels for all poliovirus types in all study groups, with no significant differences between the study groups. Conclusion The decay of poliovirus antibodies over a 21–22-month period was similar regardless of the type of booster vaccine used, suggesting the scientific data of Salk IPV long-term persistence and decay may be broadly applicable to Sabin IPV. PMID:27066157

  6. Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut.

    PubMed

    Zhang, S; Racaniello, V R

    1997-07-01

    Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.

  7. Pentavalent replicon vaccines against botulinum neurotoxins and tetanus toxin using DNA-based Semliki Forest virus replicon vectors

    PubMed Central

    Yu, YunZhou; Liu, Si; Ma, Yao; Gong, Zheng-Wei; Wang, Shuang; Sun, Zhi-Wei

    2014-01-01

    The clostridial neurotoxin (CNT) family includes botulinum neurotoxin (BoNT), serotypes A, B, E, and F of which can cause human botulism, and tetanus neurotoxin (TeNT), which is the causative agent of tetanus. This suggests that the greatest need is for a multivalent or multiagent vaccine that provides protection against all 5 agents. In this study, we investigated the feasibility of generating several pentavalent replicon vaccines that protected mice against BoNTs and TeNT. First, we evaluated the potency of individual replicon DNA or particle vaccine against TeNT, which induced strong antibody and protective responses in BALB/c mice following 2 or 3 immunizations. Then, the individual replicon TeNT vaccines were combined with tetravalent BoNTs vaccines to prepare 4 types of pentavalent replicon vaccines. These replicon DNA or particle pentavalent vaccines could simultaneously and effectively induce antibody responses and protect effects against the 5 agents. Finally, a solid-phase assay showed that the sera of pentavalent replicon formulations-immunized mice inhibited the binding of THc to the ganglioside GT1b as the sera of individual replicon DNA or particle-immunized mice. These results indicated these pentavalent replicon vaccines could protect against the 4 BoNT serotypes and effectively neutralize and protect the TeNT. Therefore, our studies demonstrate the utility of combining replicon DNA or particle vaccines into multi-agent formulations as potent pentavalent vaccines for eliciting protective responses against BoNTs and TeNT. PMID:25424795

  8. Rapid diagnosis of poliovirus infection by PCR amplification.

    PubMed Central

    Chezzi, C

    1996-01-01

    A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections. PMID:8784577

  9. Cell-Free, De Nova Synthesis of Poliovirus

    NASA Astrophysics Data System (ADS)

    Molla, Akhteruzzaman; Paul, Aniko V.; Wimmer, Eckard

    1991-12-01

    Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.

  10. Transgenic mice carrying the human poliovirus receptor: new animal models for study of poliovirus neurovirulence.

    PubMed Central

    Horie, H; Koike, S; Kurata, T; Sato-Yoshida, Y; Ise, I; Ota, Y; Abe, S; Hioki, K; Kato, H; Taya, C

    1994-01-01

    Recombinant viruses between the virulent Mahoney and attenuated Sabin 1 strains of poliovirus type 1 were subjected to neurovirulence tests using a transgenic (Tg) mouse line, ICR-PVRTg1, that carried the human poliovirus receptor gene. The Tg mice were inoculated intracerebrally with these recombinant viruses and observed for clinical signs, histopathological lesions, and viral antigens as parameters of neurovirulence of the viruses. These parameters observed in the Tg mice were different for different inoculated viruses. Dose-dependent incidences of paralysis and of death were observed in the Tg mice inoculated with any viruses used. This indicates that values of 50% lethal dose are useful to score a wide range of neurovirulence of poliovirus. The neurovirulence of individual viruses estimated by the Tg mouse model had a strong correlation with those estimated by monkey model. Consequently, the mouse tests identified the neurovirulence determinants on the genome of poliovirus that had been identified by monkey tests. In addition, the mouse tests revealed new neurovirulence determinants, that is, different nucleotides between the two strains at positions 189 and 21 and/or 935 in the 5'-proximal 1,122 nucleotides. The Tg mice used in this study may be suitable for replacing monkeys for investigating poliovirus neurovirulence. Images PMID:8289371

  11. Poliovirus tropism and attenuation are determined after internal ribosome entry

    PubMed Central

    Kauder, Steven E.; Racaniello, Vincent R.

    2004-01-01

    Poliovirus replication is limited to a few organs, including the brain and spinal cord. This restricted tropism may be a consequence of organ-specific differences in translation initiation by the poliovirus internal ribosome entry site (IRES). A C-to-U mutation at base 472 in the IRES of the Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence, may further restrict tropism by eliminating viral replication in the CNS. To determine the relationship between IRES-mediated translation and poliovirus tropism, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The IRESs of poliovirus, the cardiotropic coxsackievirus B3 (CVB3), and the hepatotropic hepatitis C virus (HCV) mediate translation in many organs, including those that do not support viral replication. A translation defect associated with the Sabin type 3 IRES was observed in all organs examined. Poliovirus type 1 and recombinant polioviruses dependent on the IRES of CVB3 or HCV replicate in the CNS of mice and cause paralysis. Although the type 3 Sabin strain is an effective vaccine, polioviruses with a U at base 472 of the IRES cause paralysis in newborn mice. Tropism of wild-type and vaccine strains of poliovirus is therefore determined after internal ribosome entry. PMID:15199409

  12. Naked singularities as particle accelerators. II

    SciTech Connect

    Patil, Mandar; Joshi, Pankaj S.; Malafarina, Daniele

    2011-03-15

    We generalize here our earlier results on particle acceleration by naked singularities. We showed recently [M. Patil and P. S. Joshi, Phys. Rev. D 82, 104049 (2010).] that the naked singularities that form due to the gravitational collapse of massive stars provide a suitable environment where particles could get accelerated and collide at arbitrarily high center-of-mass energies. However, we focused there only on the spherically symmetric gravitational collapse models, which were also assumed to be self-similar. In this paper, we broaden and generalize the result to all gravitational collapse models leading to the formation of a naked singularity as the final state of collapse, evolving from a regular initial data, without making any prior restrictive assumptions about the spacetime symmetries such as above. We show that, when the particles interact and collide near the Cauchy horizon, the energy of collision in the center-of-mass frame will be arbitrarily high, thus offering a window to the Planck scale physics. We also consider the issue of various possible physical mechanisms of generation of such very high-energy particles from the vicinity of naked singularity. We then construct a model of gravitational collapse to a timelike naked singularity to demonstrate the working of these ideas, where the pressure is allowed to be negative, but the energy conditions are respected. We show that a finite amount of mass-energy density has to be necessarily radiated away from the vicinity of the naked singularity as the collapse evolves. Therefore, the nature of naked singularities, both at the classical and quantum level, could play an important role in the process of particle acceleration, explaining the occurrence of highly energetic outgoing particles in the vicinity of the Cauchy horizon that participate in extreme high-energy collisions.

  13. Gravitational lensing by rotating naked singularities

    SciTech Connect

    Gyulchev, Galin N.; Yazadjiev, Stoytcho S.

    2008-10-15

    We model massive compact objects in galactic nuclei as stationary, axially symmetric naked singularities in the Einstein-massless scalar field theory and study the resulting gravitational lensing. In the weak deflection limit we study analytically the position of the two weak field images, the corresponding signed and absolute magnifications as well as the centroid up to post-Newtonian order. We show that there are static post-Newtonian corrections to the signed magnification and their sum as well as to the critical curves, which are functions of the scalar charge. The shift of the critical curves as a function of the lens angular momentum is found, and it is shown that they decrease slightly for the weakly naked and vastly for the strongly naked singularities with the increase of the scalar charge. The pointlike caustics drift away from the optical axis and do not depend on the scalar charge. In the strong deflection limit approximation, we compute numerically the position of the relativistic images and their separability for weakly naked singularities. All of the lensing quantities are compared to particular cases as Schwarzschild and Kerr black holes as well as Janis-Newman-Winicour naked singularities.

  14. Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

    PubMed Central

    Cheng, H P; Lessie, T G

    1994-01-01

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates. Images PMID:7517389

  15. Guidance for Assessment of Poliovirus Vaccination Status and Vaccination of Children Who Have Received Poliovirus Vaccine Outside the United States.

    PubMed

    Marin, Mona; Patel, Manisha; Oberste, Steve; Pallansch, Mark A

    2017-01-13

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis (polio). Since then, wild poliovirus (WPV) cases have declined by >99.9%, from an estimated 350,000 cases of polio each year to 74 cases in two countries in 2015 (1). This decrease was achieved primarily through the use of trivalent oral poliovirus vaccine (tOPV), which contains types 1, 2, and 3 live, attenuated polioviruses. Since 2000, the United States has exclusively used inactivated polio vaccine (IPV), which contains all three poliovirus types (2,3). In 2013, the World Health Organization (WHO) set a target of a polio-free world by 2018 (4). Of the three WPV types, type 2 was declared eradicated in September 2015. To remove the risk for infection with circulating type 2 vaccine-derived polioviruses (cVDPV), which can lead to paralysis similar to that caused by WPV, all OPV-using countries simultaneously switched in April 2016 from tOPV to bivalent OPV (bOPV), which contains only types 1 and 3 polioviruses (5). This report summarizes current Advisory Committee on Immunization Practices (ACIP) recommendations for poliovirus vaccination and provides CDC guidance, in the context of the switch from tOPV to bOPV, regarding assessment of vaccination status and vaccination of children who might have received poliovirus vaccine outside the United States, to ensure that children living in the United States (including immigrants and refugees) are protected against all three poliovirus types. This guidance is not new policy and does not change the recommendations of ACIP for poliovirus vaccination in the United States. Children living in the United States who might have received poliovirus vaccination outside the United States should meet ACIP recommendations for poliovirus vaccination, which require protection against all three poliovirus types by age-appropriate vaccination with IPV or tOPV. In the absence of vaccination records indicating receipt of these vaccines, only vaccination or

  16. Interrupting the transmission of wild polioviruses with vaccines: immunological considerations.

    PubMed Central

    Ghendon, Y.; Robertson, S. E.

    1994-01-01

    In 1988 the World Health Assembly set the goal of global poliomyelitis eradication by the year 2000. Substantial progress has been made, and 143 countries reported no poliomyelitis cases associated with the wild virus in 1993. This article reviews the immunological considerations relevant to interrupting the transmission of wild polioviruses with vaccines. Although serum immunity prevents poliomyelitis in the individual, it is local immunity that is important in preventing the transmission of polioviruses in the community. Natural infection and vaccination with oral polioviruses vaccine (OPV) produce local immunity in the intestine and the nasopharynx in about 70-80% of individuals. In contrast, inactivated poliovirus vaccine (IPV) produces local intestinal immunity in only 20-30% of the individuals. With either vaccine, however, a substantial proportion of the immunized population can transmit the wild virus. Moreover, although serum immunity is long-lasting, limited data suggest that local immunity may not be as persistent. To interrupt the transmission of wild polioviruses efforts should be made to achieve and sustain high levels of poliovirus vaccine coverage. Recent outbreaks show that wild poliovirus poses a risk for unimmunized individuals, even when overall coverage levels are high. Delivery of poliovirus vaccine to hard-to-reach populations will be of increasing importance as countries progress toward the final stages of poliomyelitis eradication. The immunization status of persons from poliomyelitis-free countries should be updated prior to travel to poliomyelitis-endemic areas. PMID:7867144

  17. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., some of these reagents consist of poliovirus antisera conjugated with a fluorescent...

  18. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., some of these reagents consist of poliovirus antisera conjugated with a fluorescent...

  19. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., some of these reagents consist of poliovirus antisera conjugated with a fluorescent...

  20. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., some of these reagents consist of poliovirus antisera conjugated with a fluorescent...

  1. 21 CFR 866.3405 - Poliovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Poliovirus serological reagents. 866.3405 Section 866.3405 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES..., some of these reagents consist of poliovirus antisera conjugated with a fluorescent...

  2. Environmental Isolation of Circulating Vaccine-Derived Poliovirus After Interruption of Wild Poliovirus Transmission - Nigeria, 2016.

    PubMed

    Etsano, Andrew; Damisa, Eunice; Shuaib, Faisal; Nganda, Gatei Wa; Enemaku, Ogu; Usman, Samuel; Adeniji, Adekunle; Jorba, Jaume; Iber, Jane; Ohuabunwo, Chima; Nnadi, Chimeremma; Wiesen, Eric

    2016-08-05

    In September 2015, more than 1 year after reporting its last wild poliovirus (WPV) case in July 2014 (1), Nigeria was removed from the list of countries with endemic poliovirus transmission,* leaving Afghanistan and Pakistan as the only remaining countries with endemic WPV. However, on April 29, 2016, a laboratory-confirmed, circulating vaccine-derived poliovirus type 2 (cVDPV2) isolate was reported from an environmental sample collected in March from a sewage effluent site in Maiduguri Municipal Council, Borno State, a security-compromised area in northeastern Nigeria. VDPVs are genetic variants of the vaccine viruses with the potential to cause paralysis and can circulate in areas with low population immunity. The Nigeria National Polio Emergency Operations Center initiated emergency response activities, including administration of at least 2 doses of oral poliovirus vaccine (OPV) to all children aged <5 years through mass campaigns; retroactive searches for missed cases of acute flaccid paralysis (AFP), and enhanced environmental surveillance. Approximately 1 million children were vaccinated in the first OPV round. Thirteen previously unreported AFP cases were identified. Enhanced environmental surveillance has not resulted in detection of additional VDPV isolates. The detection of persistent circulation of VDPV2 in Borno State highlights the low population immunity, surveillance limitations, and risk for international spread of cVDPVs associated with insurgency-related insecurity. Increasing vaccination coverage with additional targeted supplemental immunization activities and reestablishment of effective routine immunization activities in newly secured and difficult-to-reach areas in Borno is urgently needed.

  3. Construction and applications of rabbit hemorrhagic disease virus replicon.

    PubMed

    Wang, Binbin; Zhe, Mingjia; Chen, Zongyan; Li, Chuanfeng; Meng, Chunchun; Zhang, Miaotao; Liu, Guangqing

    2013-01-01

    The study of rabbit hemorrhagic disease virus (RHDV) has long been hindered by the absence of an in vitro culture system. In this study, using RHDV as a model, a series of DNA-based reporter replicons were constructed in which the firefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon. In this construct, the Fluc gene was inserted where the coding region of viral structural protein was deleted and was under the control of a minimal cytomegalovirus (CMV) immediate-early promoter. Fluc activity analysis showed that these reporter replicons replicate efficiently in mammalian cells. On the basis of the replicon, 5'non-coding regions (5'NCR) and genome-linked protein (VPg) were deleted, and the effect on the expression of replicon was analyzed. The results showed that the expression level of Fluc was reduced in the absence of 5'NCR and VPg, suggesting that the 5'NCR and VPg may play an important role in replication and/or translation of RHDV. To further verify the speculation, we also constructed a replication deficient mutant (pRHDV-luc/Δ3D), and the impact of 5'NCR and VPg deletion on viral translation efficiency was analyzed, our results indicated that both VPg and 5'NCR were involved in RHDV translation.

  4. Bioinformatics analysis and genetic diversity of the poliovirus.

    PubMed

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Wang, Shujing; Xu, Ruixue

    2014-12-01

    Poliomyelitis, a disease which can manifest as muscle paralysis, is caused by the poliovirus, which is a human enterovirus and member of the family Picornaviridae that usually transmits by the faecal-oral route. The viruses of the OPV (oral poliovirus attenuated-live vaccine) strains can mutate in the human intestine during replication and some of these mutations can lead to the recovery of serious neurovirulence. Informatics research of the poliovirus genome can be used to explain further the characteristics of this virus. In this study, sequences from 100 poliovirus isolates were acquired from GenBank. To determine the evolutionary relationship between the strains, we compared and analysed the sequences of the complete poliovirus genome and the VP1 region. The reconstructed phylogenetic trees for the complete sequences and the VP1 sequences were both divided into two branches, indicating that the genetic relationships of the whole poliovirus genome and the VP1 sequences are very similar. This branching indicates that the virulence and pathogenicity of poliomyelitis may be associated with the VP1 region. Sequence alignment of the VP1 region revealed numerous mutation sites in which mutation rates of >30 % were detected. In a group of strains recorded in the USA, mutation sites and mutation types were the same and this may be associated with their distribution in the evolutionary tree and their genetic relationship. In conclusion, the genetic evolutionary relationships of poliovirus isolate sequences are determined to a great extent by the VP1 protein, and poliovirus strains located on the same branch of the phylogenetic tree contain the same mutation spots and mutation types. Hence, the genetic characteristics of the VP1 region in the poliovirus genome should be analysed to identify the transmission route of poliovirus and provide the basis of viral immunity development.

  5. Interruption of poliovirus transmission in Ghana: molecular epidemiology of wild-type 1 poliovirus isolated from 1995 to 2008.

    PubMed

    Odoom, John Kofi; Forrest, Lindsay; Dunn, Glynis; Osei-Kwasi, Mubarak; Obodai, Evangeline; Arthur-Quarm, Jacob; Barnor, Jacob; Minor, Philip D; Martin, Javier

    2012-10-01

    Described in detail is the molecular epidemiology of wild-type 1 poliovirus circulation in Ghana between 1995-2008, following the implementation of a surveillance system for cases of acute flaccid paralysis and poliovirus infection. Molecular phylogenetic analysis combined with a detailed evaluation of epidemiological indicators revealed that the geographical and temporal circulation of wild-type poliovirus in Ghana was determined by the quality of the implementation of global eradication strategies. The transmission of "indigenous" wild-type 1 poliovirus was eliminated in 1999. However, a drastic reduction in national immunization campaigns resulted in the importation in 2003 and 2008 of wild-type 1 poliovirus from neighboring countries. Both outbreaks were promptly interrupted following resumption of immunization activities. The results detailed here provide scientific evidence that supports the feasibility of polio eradication in Central West Africa, one of the remaining endemic areas for the disease, provided that comprehensive immunization campaigns and sensitive surveillance systems are in place.

  6. The Optimum Thermal Environment for Naked Babies

    PubMed Central

    Hey, E. N.; Katz, G.

    1970-01-01

    The optimum thermal environment in which to nurse a baby naked in an incubator has been defined from a knowledge of the magnitude of the factors affecting thermal balance. Such a neutral environment allows body temperature to remain normal while oxygen consumption and evaporative water loss are both at a minimum. PMID:5427846

  7. Poliovirus aggregates and their survival in water.

    PubMed Central

    Young, D C; Sharp, D G

    1977-01-01

    Inactivation of aggregated poliovirus by bromine is characterized by a continuously decreasing reaction rate. Poliovirus released from infected cells in these experiments by alternate freezing and thawing in water without electrolytes has always been aggregated. The aggregates persist even on 7,000-fold dilution in ion-free water. Virus similarly released into phosphate-buffered saline solution may be well dispersed, but it aggregates when sedimented into a salt-free sucrose gradient or when it is diluted as little as 10-fold in water. Large one-step dilutions of dispersed virus in water remain dispersed. Aggregated virus was not dispersed by one-step dilution (7,000-fold) in distilled or untreated lake water but was dispersed if phosphate-buffered saline or clarified secondary sewage plant effluent was used as diluent. Dispersed virus aggregates at all dilutions in alum-treated, finished water from the city filter plant. This may be the result of complex formation with insoluble material rather than virion-virion aggregation. A simple procedure is described for rendering a very dilute suspension of mixed virion aggregates into a three-part spectrum of sizes. Images PMID:189686

  8. Circulating vaccine-derived polioviruses: current state of knowledge.

    PubMed Central

    Kew, Olen M.; Wright, Peter F.; Agol, Vadim I.; Delpeyroux, Francis; Shimizu, Hiroyuki; Nathanson, Neal; Pallansch, Mark A.

    2004-01-01

    Within the past 4 years, poliomyelitis outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) have occurred in Hispaniola (2000-01), the Philippines (2001), and Madagascar (2001-02). Retrospective studies have also detected the circulation of endemic cVDPV in Egypt (1988-93) and the likely localized spread of oral poliovirus vaccine (OPV)-derived virus in Belarus (1965-66). Gaps in OPV coverage and the previous eradication of the corresponding serotype of indigenous wild poliovirus were the critical risk factors for all cVDPV outbreaks. The cVDPV outbreaks were stopped by mass immunization campaigns using OPV. To increase sensitivity for detecting vaccine-derived polioviruses (VDPVs), in 2001 the Global Polio Laboratory Network implemented additional testing requirements for all poliovirus isolates under investigation. This approach quickly led to the recognition of the Philippines and Madagascar cVDPV outbreaks, but of no other current outbreaks. The potential risk of cVDPV emergence has increased dramatically in recent years as wild poliovirus circulation has ceased in most of the world. The risk appears highest for the type 2 OPV strain because of its greater tendency to spread to contacts. The emergence of cVDPVs underscores the critical importance of eliminating the last pockets of wild poliovirus circulation, maintaining universally high levels of polio vaccine coverage, stopping OPV use as soon as it is safely possible to do so, and continuing sensitive poliovirus surveillance into the foreseeable future. Particular attention must be given to areas where the risks for wild poliovirus circulation have been highest, and where the highest rates of polio vaccine coverage must be maintained to suppress cVDPV emergence. PMID:15106296

  9. FMDV replicons encoding green fluorescent protein are replication competent.

    PubMed

    Tulloch, Fiona; Pathania, Uday; Luke, Garry A; Nicholson, John; Stonehouse, Nicola J; Rowlands, David J; Jackson, Terry; Tuthill, Toby; Haas, Juergen; Lamond, Angus I; Ryan, Martin D

    2014-12-01

    The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

  10. Identification of two replicons in phage-plasmid P4.

    PubMed

    Tocchetti, A; Serina, S; Terzano, S; Dehò, G; Ghisotti, D

    1998-06-05

    DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

  11. Poliovirus sampling by using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips.

    PubMed

    Maes, Piet; Van Doren, Els; Denys, Barbara; Thoelen, Inge; Rahman, Mustafizur; Vijgen, Leen; Van Ranst, Marc

    2004-12-17

    To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.

  12. Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle.

    PubMed

    Kamrud, K I; Alterson, K; Custer, M; Dudek, J; Goodman, C; Owens, G; Smith, J F

    2010-07-01

    Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

  13. Effect of formaldehyde inactivation on poliovirus.

    PubMed

    Wilton, Thomas; Dunn, Glynis; Eastwood, David; Minor, Philip D; Martin, Javier

    2014-10-01

    Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells. Importance: This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing of

  14. Effect of Formaldehyde Inactivation on Poliovirus

    PubMed Central

    Dunn, Glynis; Eastwood, David; Minor, Philip D.; Martin, Javier

    2014-01-01

    ABSTRACT Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells. IMPORTANCE This study represents a novel insight into the molecular mechanisms responsible for poliovirus inactivation. We show that inactivation with formaldehyde has an effect on early steps of viral replication as it reduces the ability of PV to bind to hPVR, decreases the sensitivity of PV to convert to 135S particles, and abolishes the infectivity of its viral RNA. These changes are likely responsible for the loss of infectivity shown by PV following inactivation. Techniques used in this study represent new approaches for the characterization of inactivated PV products and could be useful in developing improved methods for the production and quality control testing

  15. Poliovirus protein 2BC increases cytosolic free calcium concentrations.

    PubMed Central

    Aldabe, R; Irurzun, A; Carrasco, L

    1997-01-01

    Poliovirus-infected cells undergo an increase in cytoplasmic calcium concentrations from the 4th h postinfection. The protein responsible for this effect was identified by the expression of different poliovirus nonstructural proteins in HeLa cells by using a recombinant vaccinia virus system. Synthesis of protein 2BC enhances cytoplasmic calcium concentrations in a manner similar to that observed in poliovirus-infected cells. To identify the regions in 2BC involved in modifying cytoplasmic calcium levels, several 2BC variants were generated. Regions present in both 2B and 2C are necessary to augment cellular free calcium levels. Therefore, in addition to inducing proliferation of membranous vesicles, poliovirus protein 2BC also alters cellular calcium homeostasis. PMID:9223520

  16. Poliovirus protease 3C(pro) kills cells by apoptosis.

    PubMed

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation. Copyright 2000 Academic Press.

  17. Identification and Analysis of Antiviral Compounds Against Poliovirus.

    PubMed

    Leyssen, Pieter; Franco, David; Tijsma, Aloys; Lacroix, Céline; De Palma, Armando; Neyts, Johan

    2016-01-01

    The Global Polio Eradication Initiative, launched in 1988, had as its goal the eradication of polio worldwide by the year 2000 through large-scale vaccinations campaigns with the live attenuated oral PV vaccine (OPV) (Griffiths et al., Biologicals 34:73-74, 2006). Despite substantial progress, polio remains endemic in several countries and new imported cases are reported on a regular basis ( http://www.polioeradication.org/casecount.asp ).It was recognized by the poliovirus research community that developing antivirals against poliovirus would be invaluable in the post-OPV era. Here, we describe three methods essential for the identification of selective inhibitors of poliovirus replication and for determining their mode of action by time-of-drug-addition studies as well as by the isolation of compound-resistant poliovirus variants.

  18. Gravitational radiation from a cylindrical naked singularity

    SciTech Connect

    Nakao, Ken-ichi; Morisawa, Yoshiyuki

    2005-06-15

    We construct an approximate solution which describes the gravitational emission from a naked singularity formed by the gravitational collapse of a cylindrical thick shell composed of dust. The assumed situation is that the collapsing speed of the dust is very large. In this situation, the metric variables are obtained approximately by a kind of linear perturbation analysis in the background Morgan solution which describes the motion of cylindrical null dust. The most important problem in this study is what boundary conditions for metric and matter variables should be imposed at the naked singularity. We find a boundary condition that all the metric and matter variables are everywhere finite at least up to the first order approximation. This implies that the spacetime singularity formed by this high-speed dust collapse is very similar to that formed by the null dust and the final singularity will be a conical one. Weyl curvature is completely released from the collapsed dust.

  19. NAKE'MUU: VILLAGE ON THE EDGE

    SciTech Connect

    B. VIERRA; ET AL

    2001-02-01

    Pursuant to federal requirements under the National Environmental Policy Act, in 1996 the Department of Energy (DOE) completed an environmental impact statement, record of decision, and mitigation action plan (MAP) for the Dual Axis Radiographic Hydrodynamic Test (DARHT) Facility. DARHT is a new explosives testing facility located at TA-15, Los Alamos National Laboratory (LANL). As part of the implementation of the MAP, the ESH-20 Cultural Resources Team at LANL is conducting a long-term monitoring program at the ancestral pueblo site of Nake'muu (LA 12655). DOE considers the monitoring program to be an appropriate and necessary mitigation for the potential operational impacts associated with the DARHT Facility. This chapter presents the preliminary findings of the monitoring program from 1997 through 2000. The Nake'muu site monitoring program was initiated by ESH-20 and the Mesa Verde Architectural Team, National Park Service (NPS) in 1997. Archaeologists from LANL and NPS have completed the detailed recording of all the walls at Nake'muu and the final site evaluation plan was finished in 1998 (Nordby et al. 1998). The plan describes the methods used for site monitoring, continued site revisits, and updates on wall condition by LANL archaeologists. The primary objective of the monitoring program is to identify and evaluate the long-term effects of the ambient environment and DARHT Facility operations on the architecture at Nake'muu. Is the dynamic-testing program affecting the site, and if so, to what degree? What are the short-term and long-term implications of facility operations?

  20. Naked DNA for liver gene transfer.

    PubMed

    Liu, Feng; Tyagi, Pradeep

    2005-01-01

    The majority of acquired and inherited genetic disorders, including most inborn errors of metabolism, are manifested in the liver. Therefore, it is hardly any surprise to see a large number of Medline reports describing gene therapy efforts in preclinical settings directed toward this organ (Inoue et al., 2004; Oka and Chen, 2004). Of late, non-viral vectors have garnered a lot of attention from the biomedical research community engaged in liver gene therapy (Gupta et al., 2004). However, the first initiative toward gene transfer to the liver using a non-viral approach was taken by Hickman et al. (1994), who applied the technique of naked DNA injection pioneered by Wolff (1990) for skeletal muscle. Direct injection of naked DNA resulted in low, variable and localized gene expression in the rat liver. Consequently, several developments reported in the literature since then aimed to improve hepatic gene expression by employing both surgical and nonsurgical methods. These developments include the exploitation of the unique vasculature of liver as well as the use of electric and mechanical force as an adjunct to the systemic administration of the naked plasmid gene. This chapter focuses on these developments reported from various laboratories, including ours. In addition, the underlying mechanism responsible for the dramatic increase in gene expression using these latest approaches for non-viral gene transfer to the liver is also discussed.

  1. Exploring the fitness landscape of poliovirus

    NASA Astrophysics Data System (ADS)

    Bianco, Simone; Acevedo, Ashely; Andino, Raul; Tang, Chao

    2012-02-01

    RNA viruses are known to display extraordinary adaptation capabilities to different environments, due to high mutation rates. Their very dynamical evolution is captured by the quasispecies concept, according to which the viral population forms a swarm of genetic variants linked through mutation, which cooperatively interact at a functional level and collectively contribute to the characteristics of the population. The description of the viral fitness landscape becomes paramount towards a more thorough understanding of the virus evolution and spread. The high mutation rate, together with the cooperative nature of the quasispecies, makes it particularly challenging to explore its fitness landscape. I will present an investigation of the dynamical properties of poliovirus fitness landscape, through both the adoption of new experimental techniques and theoretical models.

  2. Poliovirus: Generation, Quantification, Propagation, Purification, and Storage

    PubMed Central

    Burrill, Cecily P.; Strings, Vanessa R.; Andino, Raul

    2016-01-01

    Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (~7.5 kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non-human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented. A separate chapter concerning the generation and characterization of PV mutants will also be presented. PMID:23686830

  3. Importation and circulation of poliovirus in Bulgaria in 2001.

    PubMed Central

    Kojouharova, Mira; Zuber, Patrick L. F.; Gyurova, Snejana; Fiore, Lucia; Buttinelli, Gabriele; Kunchev, Angel; Vladimirova, Nadejda; Korsun, Neli; Filipova, Radosveta; Boneva, Roumiana; Gavrilin, Eugene; Deshpande, Jagadish M.; Oblapenko, George; Wassilak, Steven G.

    2003-01-01

    OBJECTIVE: To characterize the circumstances in which poliomyelitis occurred among three children in Bulgaria during 2001 and to describe the public health response. METHODS: Bulgarian authorities investigated the three cases of polio and their contacts, conducted faecal and serological screening of children from high-risk groups, implemented enhanced surveillance for acute flaccid paralysis, and conducted supplemental immunization activities. FINDINGS: The three cases of polio studied had not been vaccinated and lived in socioeconomically deprived areas of two cities. Four Roma children from the Bourgas district had antibody titres to serotype 1 poliovirus only, and wild type 1 virus was isolated from the faeces of two asymptomatic Roma children in the Bourgas and Sofia districts. Poliovirus isolates were related genetically and represented a single evolutionary lineage; genomic sequences were less than 90% identical to poliovirus strains isolated previously in Europe, but 98.3% similar to a strain isolated in India in 2000. No cases or wild virus isolates were found after supplemental immunization activities were launched in May 2001. CONCLUSIONS: In Bulgaria, an imported poliovirus was able to circulate for two to five months among minority populations. Surveillance data strongly suggest that wild poliovirus circulation ceased shortly after supplemental immunization activities with oral poliovirus vaccine were conducted. PMID:12973639

  4. Environmental surveillance for polioviruses in the Global Polio Eradication Initiative.

    PubMed

    Asghar, Humayun; Diop, Ousmane M; Weldegebriel, Goitom; Malik, Farzana; Shetty, Sushmitha; El Bassioni, Laila; Akande, Adefunke O; Al Maamoun, Eman; Zaidi, Sohail; Adeniji, Adekunle J; Burns, Cara C; Deshpande, Jagadish; Oberste, M Steve; Lowther, Sara A

    2014-11-01

    This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance.

  5. Poliovirus vaccination during the endgame: insights from integrated modeling.

    PubMed

    Duintjer Tebbens, Radboud J; Thompson, Kimberly M

    2017-06-01

    Managing the polio endgame requires access to sufficient quantities of poliovirus vaccines. After oral poliovirus vaccine (OPV) cessation, outbreaks may occur that require outbreak response using monovalent OPV (mOPV) and/or inactivated poliovirus vaccine. Areas covered: We review the experience and challenges with managing vaccine supplies in the context of the polio endgame. Building on models that explored polio endgame risks and the potential mOPV needs to stop outbreaks from live poliovirus reintroductions, we conceptually explore the potential demands for finished and bulk mOPV doses from a stockpile in the context of limited shelf-life of finished vaccine and time delays to convert bulk to finished vaccine. Our analysis suggests that the required size of the mOPV stockpile varies by serotype, with the highest expected needs for serotype 1 mOPV. Based on realizations of poliovirus risks after OPV cessation, the stockpile required to eliminate the chance of a stock-out appears considerably larger than the currently planned mOPV stockpiles. Expert commentary: The total required stockpile size depends on the acceptable probability of a stock-out, and increases with longer times to finish bulk doses and shorter shelf-lives of finished doses. Successful polio endgame management will require careful attention to poliovirus vaccine supplies.

  6. [Analysis of dissemination pathways for poliovirus].

    PubMed

    Ohka, Seii

    2009-06-01

    Poliomyelitis is an acute disease of the central nervous system (CNS) caused by poliovirus (PV). In humans, an infection is initiated by oral ingestion of the virus, followed by multiplication in the alimentary mucosa, from which the virus spreads through the bloodstream. Paralytic poliomyelitis initiates from the invasion of the central nervous system by circulating poliovirus, probably via the blood-brain barrier. After the virus enters the central nervous system, it replicates in neurons, especially in motor neurons, inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, a neuron-specific pathway has been reported in humans, monkeys, and PV-sensitive transgenic (Tg) mice carrying the PV receptor (hPVR/CD155) gene. It is important for the efficient virus dissemination to overcome the barriers as follows; i) to access the target tissue, ii) to enter the cells, iii) to reach the place for the replication, iv) to replicate efficiently. PV is easily transferred to humans orally; however, no rodent model for oral infections has been developed. We analyzed the each barrier above, and showed that PV is inactivated by the low pH of the gastric contents in mice. We also demonstrated that type 1 interferon signaling plays an important role in determining permissivity in the alimentary tract. As for the neural pathway, we demonstrated that direct efficient interaction between the cytoplasmic domain and cytoplasmic dynein is essential for the efficient retrograde transport of PV-containing vesicles along microtubules for the hPVR-dependent PV transport. On the other hand, we found that hPVR-independent axonal transport of PV was also observed in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist.

  7. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    PubMed

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-03-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  8. Cold-adapted poliovirus mutants bypass a postentry replication block.

    PubMed

    Dove, A W; Racaniello, V R

    1997-06-01

    In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.

  9. Genomic Characterization of Human and Environmental Polioviruses Isolated in Albania

    PubMed Central

    Divizia, Maurizio; Palombi, Leonardo; Buonomo, Ersilia; Donia, Domenica; Ruscio, Vito; Equestre, Michele; Leno, Luljeta; Panà, Augusto; Degener, Anna Marta

    1999-01-01

    Between April and December 1996, a serious outbreak of poliomyelitis occurred in Albania; almost 140 subjects were involved, and the episode presented an unusually high mortality rate (12%). During the outbreak, water samples from the Lana River in Tirana, Albania, and stool samples from two cases of paralytic poliomyelitis were collected and analyzed for the presence of polioviruses. Six polioviruses were isolated from the environmental and human samples, according to standard methods. All the samples were characterized by partial genomic sequencing of 330 bases across the 5′ untranslated region (5′-UTR) (nucleotide positions 200 to 530) and of 300 bases across the VP1 region (nucleotide positions 2474 to 2774). Comparison of these sequences with those present in data banks permitted the identification of environmental isolates Lana A and Lana B as, respectively, a Sabin-like type 2 poliovirus and an intertypic recombinant poliovirus (Sabin-like type 2/wild type 1), both bearing a G instead of an A at nucleotide position 481. The two other environmental polioviruses were similar to the isolates from the paralytic cases. They were characterized by a peculiar 5′-UTR and by a VP1 region showing 98% homology with the Albanian epidemic type 1 isolates reported by other authors. This study confirms the environmental circulation in Albania of recombinant poliovirus strains, likely sustained by a massive vaccination effort and by the presence in the environment of a type 1 poliovirus, as isolated from the Lana River in Tirana about 2 months before the first case of symptomatic acute flaccid paralysis was reported in this town. PMID:10427045

  10. The dynamic replicon: adapting to a changing cellular environment.

    PubMed

    Herrick, John

    2010-02-01

    Eukaryotic cells are often exposed to fluctuations in growth conditions as well as endogenous and exogenous stress-related agents. During development, global patterns of gene transcription change substantially, and these changes are associated with altered patterns of DNA replication and larger distances between replication origins in somatic cells compared to embryos. Conversely, when cells experience difficulties while replicating DNA, the replication program is dramatically altered and distances between replication origins decrease. Recent evidence indicates that each unit of replication, or replicon, can correspond to one or more potential replication origins, but in the case of multiple potential origins, only one is selected to initiate replication of the replicon. How one origin is selected from multiple potential origins and how origin densities are regulated during genome duplication remains unclear. The following review addresses some of the mechanisms involved in regulating replication origins during both a normal and perturbed eukaryotic cell cycle.

  11. Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

    PubMed

    Yu, Yun-Zhou; Li, Na; Ma, Yao; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-01-01

    NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity of DNA vaccines depends, to a large extent, on the presence of CpG motifs as built-in adjuvants, we addressed this issue by inserting three types of human CpG motifs (A-type, B-type, and C-type) into the backbone of nonviral DNA and viral DNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects of CpG modifications in DNA vaccines expressing three types of antigens (β-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types of CpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotype A and Bacillus anthracis A16R challenge. Taken together, these results demonstrate that insertion of human CpG motifs can differentially modulate the immunogenicity of nonviral DNA vaccines as well as viral DNA replicon vaccines. Our study provides not only a better understanding of the in vivo activities of CpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants for DNA vectors targeting specific antigens.

  12. Naked megakaryocyte nuclei: a clue to malignancy.

    PubMed

    Lefkowitz, M; Lefkowitz, E

    1977-10-01

    Bone marrow smears from 63 patients with various malignancies and a series of 51 controls were examined for the presence and percentage of naked megakaryocyte nuclei (NMN). Patients with malignancy had more than 15% NMN, which, when compared with the incidence in controls, was statistically significant. The etiology of this artifact is unknown. It is a clue to the presence of malignancy, and might be useful in following treated cases of malignancy for evidence of relapse. NMN should not be confused with metastatic malignant cells.

  13. The effects of convective overshooting on naked helium stars

    NASA Astrophysics Data System (ADS)

    Yan, Jing-Zhi; Zhu, Chun-Hua; Wang, Zhao-Jun; Lü, Guo-Liang

    2016-09-01

    Using stellar evolutionary models, we investigate the effects of convective overshooting on naked helium stars. We find that a larger value of overshooting parameter δov results in a larger convective core, which prolongs the lifetimes of naked helium stars on the helium main sequence and leads to higher effective temperatures and luminosities. For naked helium stars with masses lower than about 0.8 M⊙, they hardly become giant stars as a result of a weak burning shell. However, naked helium stars with masses between about 0.8 M⊙ and 1.1 M⊙ can evolve into giant branch phases, and finally become carbon oxygen white dwarfs.

  14. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons

    PubMed Central

    Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H.; Cardoso, M. C.

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  15. Engineered alphavirus replicon vaccines based on known attenuated viral mutants show limited effects on immunogenicity.

    PubMed

    Maruggi, Giulietta; Shaw, Christine A; Otten, Gillis R; Mason, Peter W; Beard, Clayton W

    2013-12-01

    The immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. Characterized attenuated alphavirus mutants contain changes to the genomic 5' UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction. In an attempt to create more potent replicon vaccines, we engineered a panel of Venezuelan equine encephalitis-Sindbis virus chimeric replicons that contained these attenuating mutations. Modified replicons were ranked for antigen expression and IFN induction levels in cell culture and then evaluated in mice. The results of these studies showed that differences in antigen production and IFN induction in vitro did not correlate with large changes in immunogenicity in vivo. These findings indicate that the complex interactions between innate immune response and the replicon's ability to express antigen complicate rational design of more potent alphavirus replicons.

  16. Neoteny, Prolongation of Youth: From Naked Mole Rats to "Naked Apes" (Humans).

    PubMed

    Skulachev, Vladimir P; Holtze, Susanne; Vyssokikh, Mikhail Y; Bakeeva, Lora E; Skulachev, Maxim V; Markov, Alexander V; Hildebrandt, Thomas B; Sadovnichii, Viktor A

    2017-04-01

    It has been suggested that highly social mammals, such as naked mole rats and humans, are long-lived due to neoteny (the prolongation of youth). In both species, aging cannot operate as a mechanism facilitating natural selection because the pressure of this selection is strongly reduced due to 1) a specific social structure where only the "queen" and her "husband(s)" are involved in reproduction (naked mole rats) or 2) substituting fast technological progress for slow biological evolution (humans). Lists of numerous traits of youth that do not disappear with age in naked mole rats and humans are presented and discussed. A high resistance of naked mole rats to cancer, diabetes, cardiovascular and brain diseases, and many infections explains why their mortality rate is very low and almost age-independent and why their lifespan is more than 30 years, versus 3 years in mice. In young humans, curves of mortality versus age start at extremely low values. However, in the elderly, human mortality strongly increases. High mortality rates in other primates are observed at much younger ages than in humans. The inhibition of the aging process in humans by specific drugs seems to be a promising approach to prolong our healthspan. This might be a way to retard aging, which is already partially accomplished via the natural physiological phenomenon neoteny.

  17. Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture.

    PubMed

    Yu, Mei; Peng, Betty; Chan, Katie; Gong, Ruoyu; Yang, Huiling; Delaney, William; Cheng, Guofeng

    2014-05-01

    Genotype 6 (GT6) hepatitis C virus (HCV) is prevalent in Southeast Asia and southern China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting antivirals are approved to treat GT6 HCV infection, and no cell culture systems have been described. In this study, we aimed to develop a GT6 HCV subgenomic replicon to facilitate the identification and development of new HCV therapies with pan-genotype activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an NS5A amino acid substitution (S232I) was synthesized. Electroporation of RNA encoding the GT6a replicon into Huh-7-derived cells consistently yielded 20 to 100 stable replicon colonies. Genotypic analyses of individual replicon colonies revealed new adaptive mutations across multiple viral nonstructural proteins. The E30V and K272R mutations in NS3 and the K34R mutation in NS4A were observed most frequently and were confirmed to enhance GT6a replicon replication in the presence of the NS5A amino acid substitution S232I. These new adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons for reproducible quantification of HCV replication, and the luciferase-encoding replicons enabled efficient determinations of antiviral activity for HCV inhibitors in a 384-well assay format. While nucleoside/nucleotide NS5B inhibitors and cyclophilin A inhibitors had similar antiviral activities against both GT6a and GT1b replicons, some nonnucleoside NS5B inhibitors, NS3 protease inhibitors, and NS5A inhibitors had less antiviral activity against GT6a replicons. In conjunction with other genotype replicons, this robust GT6a replicon system will aid in the development of pan-genotypic HCV regimens.

  18. [Inactivated poliovirus vaccines: an inevitable choice for eliminating poliomyelitis].

    PubMed

    Vidor, J D; Jean-Denis, Shu

    2016-12-06

    The inactivated poliovirus vaccine (IPV) is a very old tool in the fight against poliomyelitis. Though supplanted by oral poliovirus vaccine (OPV) in the 1960s and 1970s, the IPV has now become an inevitable choice because of the increasingly recognized risks associated with continuous use of OPVs. Following the pioneering work of Jonas Salk, who established key principles for the IPV, considerable experience has accumulated over the years. This work has led to modern Salk IPV-containing vaccines, based on the use of inactivated wildtype polioviruses, which have been deployed for routine use in many countries. Very good protection against paralysis is achieved with IPV through the presence of circulating antibodies able to neutralize virus infectivity toward motor neurons. In addition, with IPV, a variable degree of protection against mucosal infection (and therefore transmission) through mucosal antibodies and immune cells is achieved, depending on previous exposure of subjects to wildtype or vaccine polioviruses. The use of an IPV-followed-by-OPV sequential immunization schedule has the potential advantage of eliminating the vaccine-associated paralytic poliomyelitis (VAPP) risk, while limiting the risks of vaccine-derived poliovirus (VDPVs). Sabin strain-derived IPVs are new tools, only recently beginning to be deployed, and data are being generated to document their performance. IPVs will play an irreplaceable role in global eradication of polio.

  19. Seroepidemiology of Polioviruses among University Students in Northern Italy

    PubMed Central

    Baldo, Vincenzo; Cocchio, Silvia; Lazzari, Roberta; Saracino, Elena; Bertoncello, Chiara; Buja, Alessandra; Trevisan, Andrea

    2012-01-01

    The widespread use of poliovirus vaccination schemes has led to a marked decline in the incidence of paralytic poliomyelitis worldwide, but wild poliovirus is still endemic in some developing countries, and in 2009 a total of 23 countries reported at least 1 case of poliomyelitis caused by wild-strain polio viruses. A serological survey was thus conducted on the immunological status against polioviruses of 318 young adults, classified by their country of origin. Immunity to poliomyelitis was assessed by neutralizing antibody titration in tissues cultured on microplates. The rate of seronegativity (≤1:8) in the study population was 26.7% for poliovirus type 1, 7.2% for type 2, and 22.6% for type 3. In our sample of 318 individuals, 219 (68.9%) were Italian and 99 (31.1%) were from outside the European Union (EU). The proportion of cases found seropositive to polioviruses 1 and 3 decreased significantly with older age; this age-related decrease was more evident in the Italian group than among the non-EU subjects. Any risk of the wild virus recurring and causing paralytic poliomyelitis must be prevented, keeping Europe polio free by means of appropriate immunological protection, until polio has been conclusively eradicated all over the world. Judging from our findings, it may be worth considering administering a fifth dose of polio vaccine to adolescents. PMID:22739695

  20. Infectivity of RNA from Inactivated Poliovirus

    PubMed Central

    Nuanualsuwan, Suphachai; Cliver, Dean O.

    2003-01-01

    During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid. PMID:12620852

  1. Comprehensive screening for immunodeficiency-associated vaccine-derived poliovirus: an essential oral poliovirus vaccine cessation risk management strategy.

    PubMed

    Duintjer Tebbens, R J; Thompson, K M

    2017-01-01

    If the world can successfully control all outbreaks of circulating vaccine-derived poliovirus that may occur soon after global oral poliovirus vaccine (OPV) cessation, then immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) from rare and mostly asymptomatic long-term excretors (defined as ⩾6 months of excretion) will become the main source of potential poliovirus outbreaks for as long as iVDPV excretion continues. Using existing models of global iVDPV prevalence and global long-term poliovirus risk management, we explore the implications of uncertainties related to iVDPV risks, including the ability to identify asymptomatic iVDPV excretors to treat with polio antiviral drugs (PAVDs) and the transmissibility of iVDPVs. The expected benefits of expanded screening to identify and treat long-term iVDPV excretors with PAVDs range from US$0.7 to 1.5 billion with the identification of 25-90% of asymptomatic long-term iVDPV excretors, respectively. However, these estimates depend strongly on assumptions about the transmissibility of iVDPVs and model inputs affecting the global iVDPV prevalence. For example, the expected benefits may decrease to as low as US$260 million with the identification of 90% of asymptomatic iVDPV excretors if iVDPVs behave and transmit like partially reverted viruses instead of fully reverted viruses. Comprehensive screening for iVDPVs will reduce uncertainties and maximize the expected benefits of PAVD use.

  2. Breaking pseudo-twofold symmetry in the poliovirus 3'-UTR Y-stem by restoring Watson-Crick base pairs.

    PubMed

    Zoll, Jan; Tessari, Marco; Van Kuppeveld, Frank J M; Melchers, Willem J G; Heus, Hans A

    2007-05-01

    The previously described NMR structure of a 5'-CU-3'/5'-UU-3' motif, which is highly conserved within the 3'-UTR Y-stem of poliovirus-like enteroviruses, revealed striking regularities of the local helix geometry, thus retaining the pseudo-twofold symmetry of the RNA helix. A mutant virus with both pyrimidine base pairs changed into Watson-Crick replicated as wild type, indicating the functional importance of this symmetry relation in viral RNA replication. Here we investigated the effect of changing only one of the two pyrimidine base pairs to Watson-Crick. We determined the NMR structures of two Y-stem variants: one containing the 5'-CU-3'/5'-AU-3' motif, which has been found in wild-type virus isolates as well, and the other containing a 5'-CU-3'/5'-UG-3' motif, which is not present in any enterovirus sequenced to date. Both structures show single pyrimidine mismatches with intercalated bases. In the 5'-CU-3'/5'-AU-3' motif a C-U Watson-Crick-type base pair is formed that retains the pseudo-twofold symmetry, while in the 5'-CU-3'/5'-UG-3' motif a single asymmetric U-U mismatch breaks the twofold symmetry. Surprisingly, for the nonnatural variant no effect of the single base-pair replacement was observed on polioviral RNA replication using an in vitro replicon assay.

  3. Methods for recovering poliovirus and rotavirus from oysters.

    PubMed Central

    Speirs, J I; Pontefract, R D; Harwig, J

    1987-01-01

    Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters. PMID:2827573

  4. Annual report of the Australian National Poliovirus Reference Laboratory, 2008.

    PubMed

    Roberts, Jason A; Grant, Kristina A; Yoon, Yeon Kyung; Polychronopoulos, Sophie; Ibrahim, Aishah; Thorley, Bruce R

    2009-09-01

    The Australian National Poliovirus Reference Laboratory (NPRL) is accredited by the World Health Organization (WHO) for the testing of stool specimens from cases of acute flaccid paralysis (AFP), a major clinical presentation of poliovirus infection. The NPRL, in collaboration with the Australian Paediatric Surveillance Unit, co-ordinates surveillance for cases of AFP in children in Australia, according to criteria recommended by the WHO. Clinical specimens are referred from AFP cases in children and suspected case of poliomyelitis in persons of any age. The WHO AFP surveillance performance indicator for a polio-free country such as Australia, is 1 non-polio AFP case per 100,000 children less than 15 years of age. In 2008, the Polio Expert Committee (PEC) classified 62 cases as non-polio AFP, or 1.51 non-polio AFP cases per 100,000 children aged less than 15 years. Poliovirus infection is confirmed by virus culture of stool specimens from AFP cases as other conditions that present with acute paralysis can mimic polio. While no poliovirus was reported in Australia from any source in 2008, the non-polio enteroviruses echovirus 25, coxsackievirus B2 and echovirus 11 were isolated from stool specimens of AFP cases. The last report of a wild poliovirus in Australia was due to an importation from Pakistan in 2007. With 4 countries remaining endemic for poliomyelitis--Afghanistan, India, Nigeria and Pakistan--and more than 1,600 confirmed cases of wild poliovirus infection in 18 countries in 2008, Australia continues to be at risk of further importation events.

  5. [Precipitation antibodies against poliovirus in patients with poliomyelitis].

    PubMed

    Ruiz-Gómez, J; Bustamante, M E

    1977-01-01

    Sera from 26 children bearing poliomyelitis caused by virus tipe I and from 47 cases with paralysis of different etiology (non-poliomyelitic) were tested against antigen of poliovirus types I, II and III with an agar-immunoprecipitation reaction. All sera with neutralizing antibodies with titres of 1:1250 gave positive results in immunodifusion tests. The percentage of positiveness decrease with lower titres. No false positive reactions were found. Immunodiffusion test with antigens of poliovirus were positive in 100% of sera from patients with poliomyelitis and only in 30% in sera from patients with non polio paralysis.

  6. Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules.

    PubMed

    Bardaji, Leire; Añorga, Maite; Ruiz-Masó, José A; Del Solar, Gloria; Murillo, Jesús

    2017-01-01

    Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

  7. Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules

    PubMed Central

    Bardaji, Leire; Añorga, Maite; Ruiz-Masó, José A.; del Solar, Gloria; Murillo, Jesús

    2017-01-01

    Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities. PMID:28243228

  8. Prolonged excretion of type-2 poliovirus from a primary immune deficient patient during the transition to a type-2 poliovirus-free world, Israel, 2016

    PubMed Central

    Weil, Merav; Shulman, Lester M; Heiman, Sophia; Stauber, Tali; Alfandari, Jacqueline; Weiss, Leah; Silberstein, Ilana; Indenbaum, Viki; Mendelson, Ella; Sofer, Danit

    2016-01-01

    Wild poliovirus type-2 has been eradicated, use of live type-2 vaccine has been terminated globally, and all type-2 polioviruses are under strict laboratory containment protocols. Re-emergence may arise from prolonged asymptomatic excretion of poliovirus by hospitalised primary immune deficient (PID) patients, as described here, through repeated exposure of close contacts to high titres of infected material. At this transition time, PID patients should be screened and hospital containment protocols updated in parallel with laboratory containment. PMID:27918258

  9. Naked Viruses That Aren't Always Naked: Quasi-Enveloped Agents of Acute Hepatitis.

    PubMed

    Feng, Zongdi; Hirai-Yuki, Asuka; McKnight, Kevin L; Lemon, Stanley M

    2014-11-01

    Historically, viruses were considered to be either enveloped or nonenveloped. However, recent work on hepatitis A virus and hepatitis E virus challenges this long-held tenet. Whereas these human pathogens are shed in feces as naked nonenveloped virions, recent studies indicate that both circulate in the blood completely masked in membranes during acute infection. These membrane-wrapped virions are as infectious as their naked counterparts, although they do not express a virally encoded protein on their surface, thus distinguishing them from conventional enveloped viruses. The absence of a viral fusion protein implies that these quasi-enveloped virions have unique mechanisms for entry into cells. Like true enveloped viruses, however, these phylogenetically distinct viruses usurp components of the host ESCRT system to hijack host cell membranes and noncytolytically exit infected cells. The membrane protects these viruses from neutralizing antibodies, facilitating dissemination within the host, whereas nonenveloped virions shed in feces are stable in the environment, allowing for epidemic transmission.

  10. Interaction of Poliovirus with Its Receptor Affords a High Level of Infectivity to the Virion in Poliovirus Infections Mediated by the Fc Receptor

    PubMed Central

    Arita, Minetaro; Horie, Hitoshi; Arita, Mineo; Nomoto, Akio

    1999-01-01

    Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcγ receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system. PMID:9882307

  11. Naked shell singularities on the brane

    SciTech Connect

    Seahra, Sanjeev S.

    2005-04-15

    By utilizing nonstandard slicings of 5-dimensional Schwarzschild and Schwarzschild-AdS manifolds based on isotropic coordinates, we generate static and spherically-symmetric braneworld spacetimes containing shell-like naked null singularities. For planar slicings, we find that the brane-matter sourcing the solution is a perfect fluid with an exotic equation of state and a pressure singularity where the brane crosses the bulk horizon. From a relativistic point of view, such a singularity is required to maintain matter infinitesimally above the surface of a black hole. From the point of view of the AdS/CFT conjecture, the singular horizon can be seen as one possible quantum correction to a classical black hole geometry. Various generalizations of planar slicings are also considered for a Ricci-flat bulk, and we find that singular horizons and exotic matter distributions are common features.

  12. Airglow events visible to the naked eye

    NASA Technical Reports Server (NTRS)

    Peterson, A. W.

    1979-01-01

    During IR photographic airglow observations covering several years, three naked-eye events have been recorded. Two of these are moving luminous acoustic-gravity-wave groups of some 10-15-km wavelength, which occur near high lunar tide in the atmosphere. The events appear quickly, endure 0.5-1 h, then fade. Visible photos of two events appear enhanced, while little enhancement is present in the IR photos, although the structures are well correlated. If these events are due to OH, it is suggested that some unrecognized mechanism, perhaps a gravity wave interaction, enhances the visible transitions of the OH over the IR transitions. If the events are due to an unrecognized continuum emitter, perhaps NO, its emission must occur at the same height as the OH. Spectra seem to be the only reasonable approach to solving this problem.

  13. A naked ape would have fewer parasites.

    PubMed Central

    Pagel, Mark; Bodmer, Walter

    2003-01-01

    Unusually among the mammals, humans lack an outer layer of protective fur or hair. We propose the hypothesis that humans evolved hairlessness to reduce parasite loads, especially ectoparasites that may carry disease. We suggest that hairlessness is maintained by these naturally selected benefits and by sexual selection operating on both sexes. Hairlessness is made possible in humans owing to their unique abilities to regulate their environment via fire, shelter and clothing. Clothes and shelters allow a more flexible response to the external environment than a permanent layer of fur and can be changed or cleaned if infested with parasites. Naked molerats, another hairless and non-aquatic mammal species, also inhabit environments in which ectoparasite transmission is expected to be high, but in which temperatures are closely regulated. Our hypothesis explains features of human hairlessness-such as the marked sex difference in body hair, and its retention in the pubic regions-that are not explained by other theories. PMID:12952654

  14. Airglow events visible to the naked eye

    NASA Technical Reports Server (NTRS)

    Peterson, A. W.

    1979-01-01

    During IR photographic airglow observations covering several years, three naked-eye events have been recorded. Two of these are moving luminous acoustic-gravity-wave groups of some 10-15-km wavelength, which occur near high lunar tide in the atmosphere. The events appear quickly, endure 0.5-1 h, then fade. Visible photos of two events appear enhanced, while little enhancement is present in the IR photos, although the structures are well correlated. If these events are due to OH, it is suggested that some unrecognized mechanism, perhaps a gravity wave interaction, enhances the visible transitions of the OH over the IR transitions. If the events are due to an unrecognized continuum emitter, perhaps NO, its emission must occur at the same height as the OH. Spectra seem to be the only reasonable approach to solving this problem.

  15. Go naked: diapers affect infant walking.

    PubMed

    Cole, Whitney G; Lingeman, Jesse M; Adolph, Karen E

    2012-11-01

    In light of cross-cultural and experimental research highlighting effects of childrearing practices on infant motor skill, we asked whether wearing diapers, a seemingly innocuous childrearing practice, affects infant walking. Diapers introduce bulk between the legs, potentially exacerbating infants' poor balance and wide stance. We show that walking is adversely affected by old-fashioned cloth diapers, and that even modern disposable diapers - habitually worn by most infants in the sample - incur a cost relative to walking naked. Infants displayed less mature gait patterns and more missteps and falls while wearing diapers. Thus, infants' own diapers constitute an ongoing biomechanical perturbation while learning to walk. Furthermore, shifts in diapering practices may have contributed to historical and cross-cultural differences in infant walking.

  16. A naked ape would have fewer parasites.

    PubMed

    Pagel, Mark; Bodmer, Walter

    2003-08-07

    Unusually among the mammals, humans lack an outer layer of protective fur or hair. We propose the hypothesis that humans evolved hairlessness to reduce parasite loads, especially ectoparasites that may carry disease. We suggest that hairlessness is maintained by these naturally selected benefits and by sexual selection operating on both sexes. Hairlessness is made possible in humans owing to their unique abilities to regulate their environment via fire, shelter and clothing. Clothes and shelters allow a more flexible response to the external environment than a permanent layer of fur and can be changed or cleaned if infested with parasites. Naked molerats, another hairless and non-aquatic mammal species, also inhabit environments in which ectoparasite transmission is expected to be high, but in which temperatures are closely regulated. Our hypothesis explains features of human hairlessness-such as the marked sex difference in body hair, and its retention in the pubic regions-that are not explained by other theories.

  17. Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (nud).

    PubMed

    Taketa, S; Kikuchi, S; Awayama, T; Yamamoto, S; Ichii, M; Kawasaki, S

    2004-05-01

    To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.

  18. Hydrophobic Polycationic Coatings Disinfect Poliovirus and Rotavirus Solutions

    PubMed Central

    Larson, Alyssa M.; Hsu, Bryan B.; Rautaray, Debabrata; Haldar, Jayanta; Chen, Jianzhu

    2011-01-01

    Coating surfaces with N-alkylated polyethylenimines (PEIs), namely branched N,N-hexyl,methyl-PEI via covalent attachment to glass or linear N,N-dodecyl,methyl-PEI by physical deposition (“painting”) onto polyethylene, enables the resultant materials to quickly and efficiently disinfect aqueous solutions of (non-enveloped) poliovirus and rotavirus. PMID:20967804

  19. Stopping poliovirus vaccination after eradication: issues and challenges.

    PubMed Central

    Wood, D. J.; Sutter, R. W.; Dowdle, W. R.

    2000-01-01

    Since 1988 reported polio cases worldwide have declined by about 85% and the number of known or suspected polioendemic countries has decreased from over 120 to less than 50. With eradication of poliomyelitis approaching, issues potentially affecting when and how vaccination against poliovirus can be stopped become extremely important. Because of the potential risks and benefits inherent in such a decision, the best available science, a risk-benefit analysis, contingency plans, a stock pile of poliovirus vaccines, and the endorsement by the global policy-making committees will all be needed before vaccination can be discontinued. The scientific basis for stopping polio immunization has been reviewed by WHO. This Round Table article summarizes the current state of knowledge, provides an update on the processes and timelines for certification, containment, and stopping vaccination, and highlights some of the unanswered scientific questions that will be addressed by further research. These include whether transmission of vaccine-derived poliovirus strains could be sustained so that poliomyelitis could re-emerge in a future unvaccinated population and whether prolonged excretion of vaccine-derived poliovirus from individuals with immune deficiencies could be a mechanism through which this could occur. PMID:10812731

  20. Effects of Chlorine Concentration on the Structure of Poliovirus

    PubMed Central

    Alvarez, Maria E.; O'Brien, R. T.

    1982-01-01

    Chlorine concentrations below 0.8 mg/liter inactivated poliovirus without causing separation of the viral components. These results indicate that the release of RNA from the capsids is the result, not the cause, of virus inactivation by chlorine. PMID:6275791

  1. Hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions.

    PubMed

    Larson, Alyssa M; Hsu, Bryan B; Rautaray, Debabrata; Haldar, Jayanta; Chen, Jianzhu; Klibanov, Alexander M

    2011-03-01

    Coating surfaces with N-alkylated polyethylenimines (PEIs), namely branched N,N-hexyl,methyl-PEI via covalent attachment to glass or linear N,N-dodecyl,methyl-PEI by physical deposition ("painting") onto polyethylene, enables the resultant materials to quickly and efficiently disinfect aqueous solutions of (non-enveloped) poliovirus and rotavirus.

  2. INFECTIVITY OF RIBONUCLEIC ACID FROM POLIOVIRUS IN HUMAN CELL MONOLAYERS

    PubMed Central

    Alexander, Hattie E.; Koch, Gebhard; Mountain, Isabel Morgan; Van Damme, Olga

    1958-01-01

    Ribonucleic acid prepared by the method of Gierer and Schramm from concentrated and partially purified types I and II polioviruses has been demonstrated to be infectious for HeLa and human amnion cells in monolayers. In areas of cytopathogenic action resulting from invasion of cells by RNA, intact poliovirus, of the type from which the RNA had been prepared, is present. The infectivity of the RNA was completely inactivated by a 2 minute exposure to purified ribonuclease or to whole normal monkey serum shown to contain measurable concentrations of this enzyme. Whole virus infectivity was not influenced by RNAase or whole normal monkey serum. Normal and polio-immune globulin, desoxyribonuclease, lysozyme, proteolytic enzymes, and bovine albumin failed to inactivate the infectivity of RNA. The degree of infectivity of isolated RNA from poliovirus for cells in monolayer was greatly influenced by the ionic strength of the environment. The experimental evidence suggests that isolated poliovirus RNA is the carrier of the biological activity responsible for infection of cells and for transmission of genetic information which controls type specificity. PMID:13575680

  3. Polio eradication. Efficacy of inactivated poliovirus vaccine in India.

    PubMed

    Jafari, Hamid; Deshpande, Jagadish M; Sutter, Roland W; Bahl, Sunil; Verma, Harish; Ahmad, Mohammad; Kunwar, Abhishek; Vishwakarma, Rakesh; Agarwal, Ashutosh; Jain, Shilpi; Estivariz, Concepcion; Sethi, Raman; Molodecky, Natalie A; Grassly, Nicholas C; Pallansch, Mark A; Chatterjee, Arani; Aylward, R Bruce

    2014-08-22

    Inactivated poliovirus vaccine (IPV) is efficacious against paralytic disease, but its effect on mucosal immunity is debated. We assessed the efficacy of IPV in boosting mucosal immunity. Participants received IPV, bivalent 1 and 3 oral poliovirus vaccine (bOPV), or no vaccine. A bOPV challenge was administered 4 weeks later, and excretion was assessed 3, 7, and 14 days later. Nine hundred and fifty-four participants completed the study. Any fecal shedding of poliovirus type 1 was 8.8, 9.1, and 13.5% in the IPV group and 14.4, 24.1, and 52.4% in the control group by 6- to 11-month, 5-year, and 10-year groups, respectively (IPV versus control: Fisher's exact test P < 0.001). IPV reduced excretion for poliovirus types 1 and 3 between 38.9 and 74.2% and 52.8 and 75.7%, respectively. Thus, IPV in OPV-vaccinated individuals boosts intestinal mucosal immunity.

  4. Isolation and Characterization of Poliovirus in Cell Culture Systems.

    PubMed

    Thorley, Bruce R; Roberts, Jason A

    2016-01-01

    The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

  5. Polyadenylic acid at the 3'-terminus of poliovirus RNA.

    PubMed

    Yogo, Y; Wimmer, E

    1972-07-01

    Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.

  6. Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine.

    PubMed Central

    Alvarez, M E; O'Brien, R T

    1982-01-01

    Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis. PMID:6295277

  7. Naked singularities in higher dimensional Vaidya space-times

    SciTech Connect

    Ghosh, S. G.; Dadhich, Naresh

    2001-08-15

    We investigate the end state of the gravitational collapse of a null fluid in higher-dimensional space-times. Both naked singularities and black holes are shown to be developing as the final outcome of the collapse. The naked singularity spectrum in a collapsing Vaidya region (4D) gets covered with the increase in dimensions and hence higher dimensions favor a black hole in comparison to a naked singularity. The cosmic censorship conjecture will be fully respected for a space of infinite dimension.

  8. Mucosal and systemic adjuvant activity of alphavirus replicon particles

    NASA Astrophysics Data System (ADS)

    Thompson, Joseph M.; Whitmore, Alan C.; Konopka, Jennifer L.; Collier, Martha L.; Richmond, Erin M. B.; Davis, Nancy L.; Staats, Herman F.; Johnston, Robert E.

    2006-03-01

    Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines. vaccine vector | Venezuelan equine encephalitis virus | viral immunology | RNA virus

  9. Parental attitudes toward multiple poliovirus injections following a provider recommendation.

    PubMed

    Kolasa, M S; Bisgard, K M; Prevots, D R; Desai, S N; Dibling, K

    2001-01-01

    Changes to the polio vaccination schedule, first to a sequential inactivated poliovirus/oral poliovirus (IPV/OPV) schedule in 1996 and most recently to an all-IPV schedule, require infants to receive additional injections. Some surveys show parental hesitation concerning extra injections, whereas others show that parents prefer multiple simultaneous injections over extra immunization visits. This study describes parental behavior and attitudes about the poliovirus vaccine recommendations and additional injections at the 2- and 4-month immunization visits. Beginning July 1, 1996, providers in eight public health clinics in Cobb and Douglas Counties, Georgia, informed parents of polio vaccination options and recommended the IPV/OPV sequential schedule. A cross-sectional clinic exit survey was conducted from July 15, 1996, to January 31, 1997, with parents whose infants (younger than 6 months) were eligible for a first poliovirus vaccination. Of approximately 405 eligible infants, parents of 293 infants were approached for an interview, and 227 agreed to participate. Of those 227 participants, 210 (92%) parents chose IPV for their infant and 17 (8%) chose OPV. Of greatest concern to most parents was vaccine-associated paralytic polio (VAPP) (155, or 68.3%); the next greatest concern was an extra injection (22, or 9.7%). These parental concerns were unrelated to the number of injections the infant actually received. After receiving information on polio vaccination options and a provider recommendation, parents overwhelmingly chose IPV over OPV. Concern about VAPP was more common than objection to an extra injection. The additional injection that results from using IPV for an infant's first poliovirus vaccination appears to be acceptable to most parents.

  10. High-Throughput Next-Generation Sequencing of Polioviruses.

    PubMed

    Montmayeur, Anna M; Ng, Terry Fei Fan; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A; Oberste, M Steven; Burns, Cara C

    2017-02-01

    The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

  11. Plaque and growth characteristics of different polioviruses isolated from acute flaccid paralysis in Northern Nigeria.

    PubMed

    Sule, W F; Oyedele, O I; Osei-Kwasi, M; Odoom, J K; Adu, F D

    2008-03-01

    To determine some virulent trait-related properties of poliovirus isolates from children with acute flaccid paralysis following vaccination with oral polio vaccine (OPV). Six polioviruses earlier characterised into wild, vaccine-derived and OPV-like were studied using the plaque morphology and growth kinetics at supra-optimal temperature. Department of Virology, University of Ibadan, Nigeria. Polio isolates from six children who developed acute flaccid paralysis following vaccinations with various doses of OPV were used. All the children were located in the Northern part of the country where poliovirus is still circulating. The two vaccine-derived polioviruses acquired wild type characteristics. All the six poliovirus isolates developed different forms of plaques ranging from tiny, small and large. The plaque formed could however not be used to identify the different isolates. Growth of the different isolates at supra-optimal temperature showed that the three wild polioviruses grew to a higher titre when compared with the Sabin 2 control. The two vaccine derived isolates behaved like the wild poliovirus while the OPV-like virus acquired an intermediate characteristics between wild and sabin. The wild polioviruses represented in this study are among the last vestiges of the circulating polioviruses found in the world. It is possible that the observed biological properties of wild types 1 and 3 described in the study are typical of the West African polioviruses. These properties will provide useful previews to the final identification of some important clinical isolates especially type 1 which may grow rapidly in cell culture.

  12. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  13. Particle creation by naked singularities in higher dimensions

    SciTech Connect

    Miyamoto, Umpei; Nemoto, Hiroya; Shimano, Masahiro

    2011-04-15

    Recently, the possibility was pointed out by one of the present authors and his collaborators that an effective naked singularity referred to as ''a visible border of spacetime'' is generated by high-energy particle collision in the context of large extra dimensions or TeV-scale gravity. In this paper, we investigate the particle creation by a naked singularity in general dimensions, while adopting a model in which a marginally naked singularity forms in the collapse of a homothetic lightlike pressureless fluid. We find that the spectrum deviates from that of Hawking radiation due to scattering near the singularity but can be recast in quasithermal form. The temperature is always higher than that of Hawking radiation of a same-mass black hole, and can be arbitrarily high depending on a parameter in the model. This implies that, in principle, the naked singularity may be distinguished from a black hole in collider experiments.

  14. How to count nanoparticles with the naked eye?

    NASA Astrophysics Data System (ADS)

    Sinelnik, Artem D.; Samusev, Kirill B.

    2017-09-01

    In this work we experimentally investigated the patterns of optical Laue diffraction from photonic structures. The obtained experimental results and their interpretation allowed us to propose a very simple method of nanoparticles precise counting with the naked eye.

  15. Poly(rC) binding proteins mediate poliovirus mRNA stability.

    PubMed Central

    Murray, K E; Roberts, A W; Barton, D J

    2001-01-01

    The 5'-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882-892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124-1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5'-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5' cloverleaf RNA and rendered poliovirus mRNA unstable. A 5'-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5'-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5' cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5' cloverleaf RNA. PMID:11497431

  16. Novel hepatitis C virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a.

    PubMed

    Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi; Peng, Betty; Tian, Yang; Pagratis, Nikos; Greenstein, Andrew E; Delaney, William E

    2010-08-01

    The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (approximately 70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

  17. National choices related to inactivated poliovirus vaccine, innovation and the endgame of global polio eradication.

    PubMed

    Thompson, Kimberly M; Duintjer Tebbens, Radboud J

    2014-02-01

    Achieving the goal of a world free of poliomyelitis still requires significant effort. Although polio immunization represents a mature area, the polio endgame will require new tools and strategies, particularly as national and global health leaders coordinate the cessation of all three serotypes of oral poliovirus vaccine and increasingly adopt inactivated poliovirus vaccine (IPV). Poliovirus epidemiology and the global options for managing polioviruses continue to evolve, along with our understanding and appreciation of the resources needed and the risks that require management. Based on insights from modeling, we offer some perspective on the current status of plans and opportunities to achieve and maintain a world free of wild polioviruses and to successfully implement oral poliovirus vaccine cessation. IPV costs and potential wastage will represent an important consideration for national policy makers. Innovations may reduce future IPV costs, but the world urgently needs lower-cost IPV options.

  18. Poliovirus trafficking toward central nervous system via human poliovirus receptor-dependent and -independent pathway

    PubMed Central

    Ohka, Seii; Nihei, Coh-ichi; Yamazaki, Manabu; Nomoto, Akio

    2012-01-01

    In humans, paralytic poliomyelitis results from the invasion of the central nervous system (CNS) by circulating poliovirus (PV) via the blood–brain barrier (BBB). After the virus enters the CNS, it replicates in neurons, especially in motor neurons, inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, neural pathway has been reported in humans, monkeys, and PV-sensitive human PV receptor (hPVR/CD155)-transgenic (Tg) mice. We demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerve of hPVR-Tg mice and that intramuscularly inoculated PV causes paralysis in a hPVR-dependent manner. We also showed that hPVR-independent axonal transport of PV exists in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Circulating PV after intravenous inoculation in mice cross the BBB at a high rate in a hPVR-independent manner. We will implicate an involvement of a new possible receptor for PV to permeate the BBB based on our recent findings. PMID:22529845

  19. Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative.

    PubMed

    Xu, Wenbo; Zhang, Yong

    2016-01-01

    Stool specimens were collected from children with acute flaccid paralysis (AFP) and their contacts, and viral isolation was performed according to standard procedures. If the specimens tested positive for poliovirus, then intratypic differentiation (ITD) methods were performed on the viral isolates to determine whether the poliovirus isolates were wild or of vaccine origin, these include a poliovirus diagnostic ITD real-time PCR method and a vaccine-derived poliovirus (VDPV) screening real-time PCR method.Viral RNA was extracted from the poliovirus isolates by using the QIAamp Mini Viral RNA Extraction Kit (Qiagen) and was used for RT-PCR amplification by the standard method. The entire VP1 region of the poliovirus isolates was amplified by RT-PCR with primers that flanked the VP1-coding region. After purification of the PCR products by the QIAquick Gel Extraction Kit (Qiagen), the amplicons were bidirectionally sequenced with the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). A neurovirulence test of polioviruses isolates was carried out using PVR-Tg21 mice that expressed the human poliovirus receptor (CD155). And the temperature sensitivities of polioviruses isolates were assayed on monolayer RD cells in 24-well plates as described.

  20. A Nearly Naked Supermassive Black Hole

    NASA Astrophysics Data System (ADS)

    Condon, J. J.; Darling, Jeremy; Kovalev, Y. Y.; Petrov, L.

    2017-01-01

    During a systematic search for supermassive black holes (SMBHs) not in galactic nuclei, we identified the compact, symmetric radio source B3 1715+425 with an emission-line galaxy offset ≈ 8.5 {kpc} from the nucleus of the brightest cluster galaxy (BCG) in the redshift z = 0.1754 cluster ZwCl 8193. B3 1715+425 is too bright (brightness temperature {T}{{b}}∼ 3× {10}10 {{K}} at observing frequency ν =7.6 {GHz}) and too luminous (1.4 GHz luminosity {L}1.4{GHz}∼ {10}25 {{W}} {{Hz}}-1) to be powered by anything but an SMBH, but its host galaxy is much smaller (∼ 0.9 {kpc}× 0.6 {kpc} full width between half-maximum points) and optically fainter (R-band absolute magnitude {M}{{r}}≈ -18.2) than any other radio galaxy. Its high radial velocity {v}{{r}}≈ 1860 {km} {{{s}}}-1 relative to the BCG, continuous ionized wake extending back to the BCG nucleus, and surrounding debris indicate that the radio galaxy was tidally shredded passing through the BCG core, leaving a nearly naked SMBH fleeing from the BCG with space velocity v≳ 2000 {km} {{{s}}}-1. The radio galaxy has mass M≲ 6× {10}9 {M}ȯ and infrared luminosity {L}{IR}∼ 3× {10}11 {L}ȯ close to its dust Eddington limit, so it is vulnerable to further mass loss from radiative feedback.

  1. Naked corals: Skeleton loss in Scleractinia

    PubMed Central

    Medina, Mónica; Collins, Allen G.; Takaoka, Tori L.; Kuehl, Jennifer V.; Boore, Jeffrey L.

    2006-01-01

    Stony corals, which form the framework for modern reefs, are classified as Scleractinia (Cnidaria, Anthozoa, and Hexacorallia) in reference to their external aragonitic skeletons. However, persistent notions, collectively known as the “naked coral” hypothesis, hold that the scleractinian skeleton does not define a natural group. Three main lines of evidence have suggested that some stony corals are more closely related to one or more of the soft-bodied hexacorallian groups than they are to other scleractinians: (i) morphological similarities; (ii) lack of phylogenetic resolution in molecular analyses of scleractinians; and (iii) discrepancy between the commencement of a diverse scleractinian fossil record at 240 million years ago (Ma) and a molecule-based origination of at least 300 Ma. No molecular evidence has been able to clearly reveal relationships at the base of a well supported clade composed of scleractinian lineages and the nonskeletonized Corallimorpharia. We present complete mitochondrial genome data that provide strong evidence that one clade of scleractinians is more closely related to Corallimorpharia than it is to a another clade of scleractinians. Thus, the scleractinian skeleton, which we estimate to have originated between 240 and 288 Ma, was likely lost in the ancestry of Corallimorpharia. We estimate that Corallimorpharia originated between 110 and 132 Ma during the late- to mid-Cretaceous, coinciding with high levels of oceanic CO2, which would have impacted aragonite solubility. Corallimorpharians escaped extinction from aragonite skeletal dissolution, but some modern stony corals may not have such fortunate fates under the pressure of increased anthropogenic CO2 in the ocean. PMID:16754865

  2. Inactivation of poliovirus by gamma irradiation of wastewater sludges.

    PubMed

    Kaupert, N; Burgi, E; Scolaro, L

    1999-01-01

    The effect of gamma radiation on poliovirus infectivity seeded in sludge samples was investigated in order to determine the radiation dose required to inactivate 90% of viral infectivity (D10). Sludges were obtained from anaerobic pretreated sewages produced by San Felipe, a wastewater treatment facility located at the Tucuman province, Argentina. A D10 of 3.34 kGy was determined for poliovirus type III, Sabin strain, suspended in sludge samples. This value dropped to 1.92 kGy when the virus was suspended in water. A virucidal effect associated to sludges was also demonstrated. These results will be of interest when considering the dose of gamma radiation to be applied to wastewater sludges in order to preserve the environment from viral contamination.

  3. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    NASA Astrophysics Data System (ADS)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  4. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    SciTech Connect

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.

  5. Chlorine resistance of poliovirus isolants recovered from drinking water.

    PubMed Central

    Shaffer, P T; Metcalf, T G; Sproul, O J

    1980-01-01

    Poliovirus 1 isolants were recovered from finished drinking water produced by a modern, well-operated water treatment plant. These waters contained free chlorine residuals in excess of 1 mg/liter. The chlorine inactivation of purified high-titer preparations of two such isolants was compared with the inactivation behavior of two stock strains of poliovirus 1, LSc and Mahoney. The surviving fraction of virus derived from the two natural isolants was shown to be orders of magnitude greater than that of the standard strains. These results raise the question whether indirect drinking water standards based on free chlorine residuals are adequate public health measures, or whether direct standards based on virus determinations might be necessary. Images PMID:6257162

  6. Inactivation of poliovirus by formaldehyde: theoretical and practical aspects.

    PubMed

    GARD, S

    1957-01-01

    Since formaldehyde, either alone or in combination with other inactivating agents, is at present used in the production of all so-called "killed" poliovirus vaccines, a thorough knowledge of the kinetics of the reaction between the chemical agent and the virus, and of the mechanisms involved, is of great practical importance. In this paper the problem is discussed against the background of present knowledge of the structure of the virus and the chemical nature of the action of formaldehyde.

  7. Identification of the initiation site of poliovirus polyprotein synthesis

    SciTech Connect

    Dorner, A.J.; Dorner, L.F.; Larsen, G.R.; Wimmer, E.; Anderson, C.W.

    1982-06-01

    The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVPOO. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VPO, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.

  8. Expanding knowledge of P3 proteins in the poliovirus lifecycle

    PubMed Central

    Cameron, Craig E; Oh, Hyung Suk; Moustafa, Ibrahim M

    2010-01-01

    Poliovirus is the most extensively studied member of the order Picornavirales, which contains numerous medical, veterinary and agricultural pathogens. The picornavirus genome encodes a single polyprotein that is divided into three regions: P1, P2 and P3. P3 proteins are known to participate more directly in genome replication, for example by containing the viral RNA-dependent RNA polymerase (RdRp or 3Dpol), among several other proteins and enzymes. We will review recent data that provide new insight into the structure, function and mechanism of P3 proteins and their complexes, which are required for initiation of genome replication. Replication of poliovirus genomes occurs within macromolecular complexes, containing viral RNA, viral proteins and host-cell membranes, collectively referred to as replication complexes. P2 proteins clearly contribute to interactions with the host cell that are required for virus multiplication, including formation of replication complexes. We will discuss recent data that suggest a role for P3 proteins in formation of replication complexes. Among the least understood steps of the poliovirus lifecycle is encapsidation of genomic RNA. We will also describe data that suggest a role for P3 proteins in this step. PMID:20521933

  9. Methods for the Quality Control of Inactivated Poliovirus Vaccines.

    PubMed

    Wilton, Thomas

    2016-01-01

    Inactivated poliovirus vaccine (IPV) plays an instrumental role in the Global Poliovirus Eradication Initiative (GPEI). The quality of IPV is controlled by assessment of the potency of vaccine batches. The potency of IPV can be assessed by both in vivo and in vitro methods. In vitro potency assessment is based upon the assessment of the quantity of the D-Antigen (D-Ag) units in an IPV. The D-Ag unit is used as a measure of potency as it is largely expressed on native infectious virions and is the protective immunogen. The most commonly used in vitro test is the indirect ELISA which is used to ensure consistency throughout production.A range of in vivo assays have been developed in monkeys, chicks, guinea pigs, mice, and rats to assess the potency of IPV. All are based on assessment of the neutralizing antibody titer within the sera of the respective animal model. The rat potency test has become the favored in vivo potency test as it shows minimal variation between laboratories and the antibody patterns of rats and humans are similar. With the development of transgenic mice expressing the human poliovirus receptor, immunization-challenge tests have been developed to assess the potency of IPVs. This chapter describes in detail the methodology of these three laboratory tests to assess the quality of IPVs.

  10. Neurovirulence of Type 1 Polioviruses Isolated from Sewage in Japan

    PubMed Central

    Horie, Hitoshi; Yoshida, Hiromu; Matsuura, Kumiko; Miyazawa, Miwako; Ota, Yoshihiro; Nakayama, Takashi; Doi, Yutaka; Hashizume, So

    2002-01-01

    Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use. PMID:11772619

  11. Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

    PubMed Central

    Pata, J D; Schultz, S C; Kirkegaard, K

    1995-01-01

    Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 8 PMID:7489508

  12. Antimicrobial susceptibility and plasmid replicon typing of Salmonella enterica serovar Kentucky isolates recovered from broilers

    USDA-ARS?s Scientific Manuscript database

    Salmonella Kentucky has become the predominate serotype recovered from broiler slaughter in the United States and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serotype. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 ...

  13. Single-step construction of a picornavirus replicon RNA with precise ends.

    PubMed

    Xiong, Qing; Wang, Yuya; Xie, Bingyu; Pei, Xinyi; Peng, Yihong

    2017-10-01

    A versatile single-step method is described for constructing a picornavirus replicon RNA with precise ends to facilitate improved understanding of viral genome function and mimic native virus replication in host cells as far as possible. The key innovation in this new approach is the use of a bridge primer to both introduce a ribozyme sequence for cis-cleavage of RNA to generate precise 5' ends of EV71 RNA and also mediate overlapping assembly of two fragments. Using an EV71 replicon as a test case, precise ends for the viral replicon were shown to be important for efficient virus replication. Thus, our work provides a novel efficient way to generating higher efficient viral replicon with precise ends and this novel method can be applied to other picornaviruses' research. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus.

    PubMed

    Reap, Elizabeth A; Morris, John; Dryga, Sergey A; Maughan, Maureen; Talarico, Todd; Esch, Robert E; Negri, Sarah; Burnett, Bruce; Graham, Andrew; Olmsted, Robert A; Chulay, Jeffrey D

    2007-10-16

    We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

  15. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  16. Assessment of cell culture and polymerase chain reaction procedures for the detection of polioviruses in wastewater.

    PubMed Central

    Grabow, W. O.; Botma, K. L.; de Villiers, J. C.; Clay, C. G.; Erasmus, B.

    1999-01-01

    WHO considers that environmental surveillance for wild-type polioviruses is potentially important for surveillance for acute flaccid paralysis as a means of confirming eradication of poliomyelitis. The present study investigated methods for detecting polioviruses in a variety of water environments in South Africa. Most polioviruses were isolated on L20B mouse cells, which, however, were not selective: 16 reoviruses and 8 enteroviruses, apparently animal strains, were also isolated on these cells. Vaccine strains of polioviruses were isolated from surface waters during and shortly after two rounds of mass vaccination of children in an informal settlement where there was no sewerage. The results demonstrated the feasibility of poliovirus surveillance in such settlements. It was also evident that neither poliovirus vaccine strains nor other viruses were likely to interfere significantly with the detection of wild-type polioviruses. Optimal isolation of polioviruses was accomplished by parallel inoculation of L20B mouse cells and at least the PLC/PRF/5 human liver and buffalo green monkey (BGM) kidney cell lines. Analysis of cell cultures using the polymerase chain reaction revealed that 319 test samples contained at least 263 human enteroviruses that failed to produce a cytopathogenic effect. This type of analysis thus significantly increased the sensitivity of enterovirus detection. PMID:10680244

  17. Different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus.

    PubMed

    Tuladhar, Era; de Koning, Martijn C; Fundeanu, Irina; Beumer, Rijkelt; Duizer, Erwin

    2012-04-01

    Virucidal activity of immobilized quaternary ammonium compounds (IQACs) coated onto glass and plastic surfaces was tested against enveloped influenza A (H1N1) virus and nonenveloped poliovirus Sabin1. The IQACs tested were virucidal against the influenza virus within 2 min, but no virucidal effect against poliovirus was found in 6 h.

  18. Impact of inactivated poliovirus vaccine on mucosal immunity: implications for the polio eradication endgame

    PubMed Central

    Parker, Edward PK; Molodecky, Natalie A; Pons-Salort, Margarita; O’Reilly, Kathleen M; Grassly, Nicholas C

    2015-01-01

    The polio eradication endgame aims to bring transmission of all polioviruses to a halt. To achieve this aim, it is essential to block viral replication in individuals via induction of a robust mucosal immune response. Although it has long been recognized that inactivated poliovirus vaccine (IPV) is incapable of inducing a strong mucosal response on its own, it has recently become clear that IPV may boost immunity in the intestinal mucosa among individuals previously immunized with oral poliovirus vaccine. Indeed, mucosal protection appears to be stronger following a booster dose of IPV than oral poliovirus vaccine, especially in older children. Here, we review the available evidence regarding the impact of IPV on mucosal immunity, and consider the implications of this evidence for the polio eradication endgame. We conclude that the implementation of IPV in both routine and supplementary immunization activities has the potential to play a key role in halting poliovirus transmission, and thereby hasten the eradication of polio. PMID:26159938

  19. Managing population immunity to reduce or eliminate the risks of circulation following the importation of polioviruses.

    PubMed

    Thompson, Kimberly M; Kalkowska, Dominika A; Duintjer Tebbens, Radboud J

    2015-03-24

    Poliovirus importations into polio-free countries represent a major concern during the final phases of global eradication of wild polioviruses (WPVs). We extend dynamic transmission models to demonstrate the dynamics of population immunity out through 2020 for three countries that only used inactivated poliovirus vaccine (IPV) for routine immunization: the US, Israel, and The Netherlands. For each country, we explore the vulnerability to re-established transmission following an importation for each poliovirus serotype, including the impact of immunization choices following the serotype 1 WPV importation that occurred in 2013 in Israel. As population immunity declines below the threshold required to prevent transmission, countries become at risk for re-established transmission. Although importations represent stochastic events that countries cannot fully control because people cross borders and polioviruses mainly cause asymptomatic infections, countries can ensure that any importations die out. Our results suggest that the general US population will remain above the threshold for transmission through 2020. In contrast, Israel became vulnerable to re-established transmission of importations of live polioviruses by the late 2000s. In Israel, the recent WPV importation and outbreak response use of bivalent oral poliovirus vaccine (bOPV) eliminated the vulnerability to an importation of poliovirus serotypes 1 and 3 for several years, but not serotype 2. The Netherlands experienced a serotype 1 WPV outbreak in 1992-1993 and became vulnerable to re-established transmission in religious communities with low vaccine acceptance around the year 2000, although the general population remains well-protected from widespread transmission. All countries should invest in active management of population immunity to avoid the potential circulation of imported live polioviruses. IPV-using countries may wish to consider prevention opportunities and/or ensure preparedness for response

  20. Patients with Primary Immunodeficiencies Are a Reservoir of Poliovirus and a Risk to Polio Eradication

    PubMed Central

    Aghamohammadi, Asghar; Abolhassani, Hassan; Kutukculer, Necil; Wassilak, Steve G.; Pallansch, Mark A.; Kluglein, Samantha; Quinn, Jessica; Sutter, Roland W.; Wang, Xiaochuan; Sanal, Ozden; Latysheva, Tatiana; Ikinciogullari, Aydan; Bernatowska, Ewa; Tuzankina, Irina A.; Costa-Carvalho, Beatriz T.; Franco, Jose Luis; Somech, Raz; Karakoc-Aydiner, Elif; Singh, Surjit; Bezrodnik, Liliana; Espinosa-Rosales, Francisco J.; Shcherbina, Anna; Lau, Yu-Lung; Nonoyama, Shigeaki; Modell, Fred; Modell, Vicki; Ozen, Ahmet; Barbouche, Mohamed-Ridha; McKinlay, Mark A.

    2017-01-01

    Immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) have been isolated from primary immunodeficiency (PID) patients exposed to oral poliovirus vaccine (OPV). Patients may excrete poliovirus strains for months or years; the excreted viruses are frequently highly divergent from the parental OPV and have been shown to be as neurovirulent as wild virus. Thus, these patients represent a potential reservoir for transmission of neurovirulent polioviruses in the post-eradication era. In support of WHO recommendations to better estimate the prevalence of poliovirus excreters among PIDs and characterize genetic evolution of these strains, 635 patients including 570 with primary antibody deficiencies and 65 combined immunodeficiencies were studied from 13 OPV-using countries. Two stool samples were collected over 4 days, tested for enterovirus, and the poliovirus positive samples were sequenced. Thirteen patients (2%) excreted polioviruses, most for less than 2 months following identification of infection. Five (0.8%) were classified as iVDPVs (only in combined immunodeficiencies and mostly poliovirus serotype 2). Non-polio enteroviruses were detected in 30 patients (4.7%). Patients with combined immunodeficiencies had increased risk of delayed poliovirus clearance compared to primary antibody deficiencies. Usually, iVDPV was detected in subjects with combined immunodeficiencies in a short period of time after OPV exposure, most for less than 6 months. Surveillance for poliovirus excretion among PID patients should be reinforced until polio eradication is certified and the use of OPV is stopped. Survival rates among PID patients are improving in lower and middle income countries, and iVDPV excreters are identified more frequently. Antivirals or enhanced immunotherapies presently in development represent the only potential means to manage the treatment of prolonged excreters and the risk they present to the polio endgame. PMID:28952612

  1. An economic analysis of poliovirus risk management policy options for 2013-2052.

    PubMed

    Duintjer Tebbens, Radboud J; Pallansch, Mark A; Cochi, Stephen L; Wassilak, Steven G F; Thompson, Kimberly M

    2015-09-24

    The Global Polio Eradication Initiative plans for coordinated cessation of oral poliovirus vaccine (OPV) after interrupting all wild poliovirus (WPV) transmission, but many questions remain related to long-term poliovirus risk management policies. We used an integrated dynamic poliovirus transmission and stochastic risk model to simulate possible futures and estimate the health and economic outcomes of maintaining the 2013 status quo of continued OPV use in most developing countries compared with OPV cessation policies with various assumptions about global inactivated poliovirus vaccine (IPV) adoption. Continued OPV use after global WPV eradication leads to continued high costs and/or high cases. Global OPV cessation comes with a high probability of at least one outbreak, which aggressive outbreak response can successfully control in most instances. A low but non-zero probability exists of uncontrolled outbreaks following a poliovirus reintroduction long after OPV cessation in a population in which IPV-alone cannot prevent poliovirus transmission. We estimate global incremental net benefits during 2013-2052 of approximately $16 billion (US$2013) for OPV cessation with at least one IPV routine immunization dose in all countries until 2024 compared to continued OPV use, although significant uncertainty remains associated with the frequency of exportations between populations and the implementation of long term risk management policies. Global OPV cessation offers the possibility of large future health and economic benefits compared to continued OPV use. Long-term poliovirus risk management interventions matter (e.g., IPV use duration, outbreak response, containment, continued surveillance, stockpile size and contents, vaccine production site requirements, potential antiviral drugs, and potential safer vaccines) and require careful consideration. Risk management activities can help to ensure a low risk of uncontrolled outbreaks and preserve or further increase the

  2. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

    PubMed

    Gil-Humanes, Javier; Wang, Yanpeng; Liang, Zhen; Shan, Qiwei; Ozuna, Carmen V; Sánchez-León, Susana; Baltes, Nicholas J; Starker, Colby; Barro, Francisco; Gao, Caixia; Voytas, Daniel F

    2017-03-01

    The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

  3. Inactivation by bromine of single poliovirus particles in water.

    PubMed Central

    Floyd, R; Johnson, J D; Sharp, D G

    1976-01-01

    Quantitative electron microscopy shows that Freon-extracted poliovirus, velocity banded in a sucrose gradient, contains over 95% single particles. This well-dispersed virus reacts quite rapidly with bromine in turbulent flowing water, losing plaque titer at the rate of one log10 unit in 10s at pH 7, 2 C, and at a bromine concentration of 2.2 muM. At 10 and 20 C the rate of disinfection (log10 plaque-forming units per second) is faster, and at both temperatures it increases in approximately linear fashion with increasing bromine concentration. At 2 C such a linear relationship is not observed. Images PMID:11745

  4. Isolation of viruses from sewage, with special regard to poliovirus

    PubMed Central

    Böttiger, Margareta

    1978-01-01

    This report concerns experiments to isolate different viruses from sewage. Using a special cell-line from Utrecht, derived from human amniotic cells, it was possible to isolate poliovirus selectively when antisera against six types of coxsackievirus B were added to the tissue culture. The method was tested in connexion with the epidemiological investigation of a case of poliomyelitis in Sweden in 1977. It rapidly demonstrated that the virus implicated was present in all neighbouring sewage plants, indicating a wide distribution of the virus in the area. PMID:216501

  5. Two-Laser Interference Visible to the Naked Eye

    ERIC Educational Resources Information Center

    Kawalec, Tomasz; Bartoszek-Bober, Dobroslawa

    2012-01-01

    An experimental setup allowing the observation of two-laser interference by the naked eye is described. The key concept is the use of an electronic phase lock between two external cavity diode lasers. The experiment is suitable both for undergraduate and graduate students, mainly in atomic physics laboratories. It gives an opportunity for…

  6. Back to Basics: Naked-Eye Astronomical Observation

    ERIC Educational Resources Information Center

    Barclay, Charles

    2003-01-01

    For pupils of both sexes and all ages from about six upwards, the subject of Astronomy holds many fascinations--the rapid changes in knowledge, the large resource of available IT packages and above all the beautiful pictures from Hubble and the large Earth-based telescopes. This article, however, stresses the excitement and importance of naked-eye…

  7. Spin precession in a black hole and naked singularity spacetimes

    NASA Astrophysics Data System (ADS)

    Chakraborty, Chandrachur; Kocherlakota, Prashant; Joshi, Pankaj S.

    2017-02-01

    We propose here a specific criterion to address the existence or otherwise of Kerr naked singularities, in terms of the precession of the spin of a test gyroscope due to the frame dragging by the central spinning body. We show that there is indeed an important characteristic difference in the behavior of gyro spin precession frequency in the limit of approach to these compact objects, and this can be used, in principle, to differentiate the naked singularity from a black hole. Specifically, if gyroscopes are fixed all along the polar axis up to the horizon of a Kerr black hole, the precession frequency becomes arbitrarily high, blowing up as the event horizon is approached. On the other hand, in the case of naked singularity, this frequency remains always finite and well behaved. Interestingly, this behavior is intimately related to and is governed by the geometry of the ergoregion in each of these cases, which we analyze here. One intriguing behavior that emerges is, in the Kerr naked singularity case, the Lense-Thirring precession frequency (ΩLT ) of the gyroscope due to frame-dragging effect decreases as (ΩLT∝r ) after reaching a maximum, in the limit of r =0 , as opposed to r-3 dependence in all other known astrophysical cases.

  8. Naked at Our Age: Talking out Loud about Senior Sex

    ERIC Educational Resources Information Center

    Davis, Melanie

    2012-01-01

    "Naked at Our Age" is an excellent resource for sexually interested and/or active adults over the age of 60. The book combines the author's personal reflections, questions and stories shared by older adults, and advice from sex therapists, sexuality educators, the author, and health care providers. The breadth of topics makes the book useful to…

  9. Using Visual Interpretive Analysis in Teaching Joyce Carol Oates's "Naked."

    ERIC Educational Resources Information Center

    Brandt, Kenneth K.

    2003-01-01

    Shares some of the classroom practices that have worked well in teaching "Naked." Presents a summary of introductory comments about the story and a description of the sketching activity. Offers interpretive commentary on a selection of representative student sketches that reflect significant features of the story. Provides general…

  10. Back to Basics: Naked-Eye Astronomical Observation

    ERIC Educational Resources Information Center

    Barclay, Charles

    2003-01-01

    For pupils of both sexes and all ages from about six upwards, the subject of Astronomy holds many fascinations--the rapid changes in knowledge, the large resource of available IT packages and above all the beautiful pictures from Hubble and the large Earth-based telescopes. This article, however, stresses the excitement and importance of naked-eye…

  11. Naked at Our Age: Talking out Loud about Senior Sex

    ERIC Educational Resources Information Center

    Davis, Melanie

    2012-01-01

    "Naked at Our Age" is an excellent resource for sexually interested and/or active adults over the age of 60. The book combines the author's personal reflections, questions and stories shared by older adults, and advice from sex therapists, sexuality educators, the author, and health care providers. The breadth of topics makes the book useful to…

  12. Registration of a tufted-naked seed upland cotton germplasm

    USDA-ARS?s Scientific Manuscript database

    A tufted-naked cotton (Gossypium hirsutum L.) mutant, 9023n4t (Reg. No. GP-971, PI 667553) was developed from the cultivar SC 9023 (9023) (PI 590933) through chemical mutagenesis. Germplasm line 9023n4t was developed by the Department of Plant and Soil Science, Texas Tech University, and released in...

  13. Colorimetric "naked eye" sensing of anions in aqueous solution.

    PubMed

    Gunnlaugsson, Thorfinnur; Kruger, Paul E; Jensen, Paul; Tierney, Juliann; Ali, Haslin Dato Paduka; Hussey, Gillian M

    2005-12-23

    [structure: see text] The synthesis and UV-vis and NMR spectroscopic studies of thiourea-based colorimetric sensors for anions are presented. These sensors can recognize anions through hydrogen binding even in competitive pH-buffered aqueous solutions, giving rise to large color changes that are clearly visible to the naked eye.

  14. Two-Laser Interference Visible to the Naked Eye

    ERIC Educational Resources Information Center

    Kawalec, Tomasz; Bartoszek-Bober, Dobroslawa

    2012-01-01

    An experimental setup allowing the observation of two-laser interference by the naked eye is described. The key concept is the use of an electronic phase lock between two external cavity diode lasers. The experiment is suitable both for undergraduate and graduate students, mainly in atomic physics laboratories. It gives an opportunity for…

  15. Zebrafish Naked 1 and Naked 2 antagonize both canonical and non-canonical Wnt signaling

    PubMed Central

    Van Raay, Terence J.; Coffey, Robert J.; Solnica-Krezel, Lilianna

    2007-01-01

    Wnt signaling controls a wide range of developmental processes and its aberrant regulation can lead to disease. To better understand the regulation of this pathway, we identified zebrafish homologues of Naked Cuticle (Nkd), Nkd1 and Nkd2, which have previously been shown to inhibit canonical Wnt/β-catenin signaling. Zebrafish nkd1 expression increases substantially after the mid-blastula transition in a pattern mirroring that of activated canonical Wnt/β-catenin signaling, being expressed in both the ventrolateral blastoderm margin and also in the axial mesendoderm. In contrast, zebrafish nkd2 is maternally and ubiquitously expressed. Overexpression of Nkd1 or Nkd2a suppressed canonical Wnt/β-catenin signaling at multiple stages of early zebrafish development and also exacerbated the cyclopia and axial mesendoderm convergence and extension (C&E) defect in the non-canonical Wnt/PCP mutant silberblick (slb/wnt11). Thus, Nkds are sufficient to antagonize both canonical and non-canonical Wnt signaling. Reducing Nkd function using antisense morpholino oligonucleotides resulted in increased expression of canonical Wnt/β-catenin target genes. Finally, reducing Nkd1 function in slb mutants suppressed the axial mesendoderm C&E defect. These data indicate that zebrafish Nkd1 and Nkd2 function to limit both canonical and non-canonical Wnt signaling. PMID:17689523

  16. In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

    PubMed

    Kamrud, Kurt I; Coffield, V McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K; Berglund, Peter; Smith, Jonathan

    2010-08-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

  17. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

    PubMed Central

    diCenzo, George C.; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M.; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  18. Genome-wide profiling of S/MAR-based replicon contact sites

    PubMed Central

    Gogol-Döring, Andreas; Schreiber, Sabrina; Epplen, Jörg T.; Lipps, Hans J.

    2017-01-01

    Abstract Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites. PMID:28609784

  19. Genome-wide profiling of S/MAR-based replicon contact sites.

    PubMed

    Hagedorn, Claudia; Gogol-Döring, Andreas; Schreiber, Sabrina; Epplen, Jörg T; Lipps, Hans J

    2017-07-27

    Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Stochastic association of neighboring replicons creates replication factories in budding yeast

    PubMed Central

    Saner, Nazan; Karschau, Jens; Natsume, Toyoaki; Gierliński, Marek; Retkute, Renata; Hawkins, Michelle; Nieduszynski, Conrad A.; Blow, J. Julian; de Moura, Alessandro P.S.

    2013-01-01

    Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures. PMID:24062338

  1. Perfect fluid tori orbiting Kehagias-Sfetsos naked singularities

    NASA Astrophysics Data System (ADS)

    Stuchlík, Z.; Pugliese, D.; Schee, J.; Kučáková, H.

    2015-09-01

    We construct perfect fluid tori in the field of the Kehagias-Sfetsos (K-S) naked singularities. These are spherically symmetric vacuum solutions of the modified Hořava quantum gravity, characterized by a dimensionless parameter ω M^2, combining the gravitational mass parameter M of the spacetime with the Hořava parameter ω reflecting the role of the quantum corrections. In dependence on the value of ω M^2, the K-S naked singularities demonstrate a variety of qualitatively different behavior of their circular geodesics that is fully reflected in the properties of the toroidal structures, demonstrating clear distinction to the properties of the torii in the Schwarzschild spacetimes. In all of the K-S naked singularity spacetimes the tori are located above an "antigravity" sphere where matter can stay in a stable equilibrium position, which is relevant for the stability of the orbiting fluid toroidal accretion structures. The signature of the K-S naked singularity is given by the properties of marginally stable tori orbiting with the uniform distribution of the specific angular momentum of the fluid, l= const. In the K-S naked singularity spacetimes with ω M^2 > 0.2811, doubled tori with the same l= const can exist; mass transfer between the outer torus and the inner one is possible under appropriate conditions, while only outflow to the outer space is allowed in complementary conditions. In the K-S spacetimes with ω M^2 < 0.2811, accretion from cusped perfect fluid tori is not possible due to the non-existence of unstable circular geodesics.

  2. Polypyrimidine tract binding protein-1 (PTB1) is a determinant of the tissue and host tropism of a human rhinovirus/poliovirus chimera PV1(RIPO).

    PubMed

    Jahan, Nusrat; Wimmer, Eckard; Mueller, Steffen

    2013-01-01

    The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)'s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the

  3. Achieving high seroprevalence against polioviruses in Sri Lanka--results from a serological survey, 2014.

    PubMed

    Gamage, Deepa; Palihawadana, Paba; Mach, Ondrej; Weldon, William C; Oberste, Steven M; Sutter, Roland W

    2015-12-01

    The immunization program in Sri Lanka consistently reaches >90% coverage with oral poliovirus vaccines (OPV), and no polio supplementary vaccination campaigns have been conducted since 2003. We evaluated serological protection against polioviruses in children. A cross-sectional community-based survey was performed in three districts of Sri Lanka (Colombo, Badulla, and Killinochi). Randomly selected children in four age groups (9-11 months, 3-4 years, 7-9 years, and 15 years) were tested for poliovirus neutralizing antibodies. All 400 enrolled children completed the study. The proportion of seropositive children for poliovirus Type 1 and Type 2 was >95% for all age groups; for poliovirus Type 3 it was 95%, 90%, 77%, and 75% in the respective age groups. The vaccination coverage in our sample based on vaccination cards or parental recall was >90% in all age groups. Most Sri Lankan children are serologically protected against polioviruses through routine immunization only. This seroprevalence survey provided baseline data prior to the anticipated addition of inactivated poliovirus vaccine (IPV) into the Sri Lankan immunization program and the switch from trivalent OPV (tOPV) to bivalent OPV (bOPV).

  4. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  5. Risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication.

    PubMed

    Tebbens, Radboud J Duintjer; Pallansch, Mark A; Kew, Olen M; Cáceres, Victor M; Jafari, Hamid; Cochi, Stephen L; Sutter, Roland W; Aylward, R Bruce; Thompson, Kimberly M

    2006-12-01

    After the global eradication of wild polioviruses, the risk of paralytic poliomyelitis from polioviruses will still exist and require active management. Possible reintroductions of poliovirus that can spread rapidly in unprotected populations present challenges to policymakers. For example, at least one outbreak will likely occur due to circulation of a neurovirulent vaccine-derived poliovirus after discontinuation of oral poliovirus vaccine and also could possibly result from the escape of poliovirus from a laboratory or vaccine production facility or from an intentional act. In addition, continued vaccination with oral poliovirus vaccines would result in the continued occurrence of vaccine-associated paralytic poliomyelitis. The likelihood and impacts of reintroductions in the form of poliomyelitis outbreaks depend on the policy decisions and on the size and characteristics of the vulnerable population, which change over time. A plan for managing these risks must begin with an attempt to characterize and quantify them as a function of time. This article attempts to comprehensively characterize the risks, synthesize the existing data available for modeling them, and present quantitative risk estimates that can provide a starting point for informing policy decisions.

  6. High resolution identity testing of inactivated poliovirus vaccines

    PubMed Central

    Mee, Edward T.; Minor, Philip D.; Martin, Javier

    2015-01-01

    Background Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. Methods We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. Results All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Conclusion Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. PMID:26049003

  7. RNA binding and replication by the poliovirus RNA polymerase

    SciTech Connect

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.

  8. Cleavage sites within the poliovirus capsid protein precursors

    SciTech Connect

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.

  9. Identification and Manipulation of the Molecular Determinants Influencing Poliovirus Recombination

    PubMed Central

    Runckel, Charles; Westesson, Oscar; Andino, Raul; DeRisi, Joseph L.

    2013-01-01

    The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific “hotspots,” including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains. PMID:23408891

  10. High resolution identity testing of inactivated poliovirus vaccines.

    PubMed

    Mee, Edward T; Minor, Philip D; Martin, Javier

    2015-07-09

    Definitive identification of poliovirus strains in vaccines is essential for quality control, particularly where multiple wild-type and Sabin strains are produced in the same facility. Sequence-based identification provides the ultimate in identity testing and would offer several advantages over serological methods. We employed random RT-PCR and high throughput sequencing to recover full-length genome sequences from monovalent and trivalent poliovirus vaccine products at various stages of the manufacturing process. All expected strains were detected in previously characterised products and the method permitted identification of strains comprising as little as 0.1% of sequence reads. Highly similar Mahoney and Sabin 1 strains were readily discriminated on the basis of specific variant positions. Analysis of a product known to contain incorrect strains demonstrated that the method correctly identified the contaminants. Random RT-PCR and shotgun sequencing provided high resolution identification of vaccine components. In addition to the recovery of full-length genome sequences, the method could also be easily adapted to the characterisation of minor variant frequencies and distinction of closely related products on the basis of distinguishing consensus and low frequency polymorphisms. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

    PubMed Central

    Irurzun, A; Sánchez-Palomino, S; Novoa, I; Carrasco, L

    1995-01-01

    Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular

  12. Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

    PubMed

    Irurzun, A; Sánchez-Palomino, S; Novoa, I; Carrasco, L

    1995-12-01

    Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular

  13. Comparative analyses of extrachromosomal bacterial replicons, identification of chromids, and experimental evaluation of their indispensability.

    PubMed

    Dziewit, Lukasz; Bartosik, Dariusz

    2015-01-01

    Bacterial genomic information can be divided between various replicons, including chromosomes, plasmids, and chromids (essential plasmid-like replicons with properties of both chromosomes and plasmids). Comparative analyses of bacterial plasmids, including homology searches, phylogenetic and phylogenomic analyses, as well as network construction for the characterization of their relationships, are good starting points for the identification of chromids. Chromids possess several chromosome-like genetic features (e.g., codon usage, GC content), but most significantly, they carry housekeeping genes, which make them indispensable for cell viability. However, it is important to confirm in silico predictions experimentally. The essential nature of a predicted chromid is usually verified by the application of a target-oriented replicon curing technique, based on the incompatibility phenomenon. Further tests examining growth in various media are used to distinguish secondary chromids from plasmids, and mutational analysis (e.g., using the yeast FLP/FRT recombination system) is employed to identify essential genes carried by particular chromids.

  14. Development and preclinical evaluation of an alphavirus replicon vaccine for influenza.

    PubMed

    Hubby, Bolyn; Talarico, Todd; Maughan, Maureen; Reap, Elizabeth A; Berglund, Peter; Kamrud, Kurt I; Copp, Laura; Lewis, Whitney; Cecil, Chad; Norberg, Pamela; Wagner, Jordan; Watson, Aubrey; Negri, Sarah; Burnett, Bruce K; Graham, Andrew; Smith, Jonathan F; Chulay, Jeffrey D

    2007-11-23

    We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

  15. A cloned replicon of Saccharopolyspora phages JHJ-1 and JHJ-3 is stably maintained as a plasmid in various actinomycetes.

    PubMed

    Gaudreau, L R; Déry, C V

    1993-04-15

    A replicon of phage JHJ-1 (and JHJ-3) was cloned. The autonomously replicating phage element was maintained as a medium-copy-number shuttle plasmid in many actinomycetes, and was efficiently transmitted to spores without antibiotic selection. One gene was shown to be expressed in a vector containing the JHJ-3 replicon.

  16. Naked bodies, naked genomes: the special (but not exceptional) nature of genomic information.

    PubMed

    Sulmasy, Daniel P

    2015-05-01

    Genetic exceptionalism, the view that genomic information is different from other types of sensitive information and deserves exceptional types of protections, has been roundly criticized. However, the public still expresses special fears about the access others might have to their genomic information. In this article, it is argued that there may be a basis for the public perception that genomic information is special, even if it cannot be said that policies could or should be enacted to protect the privacy and confidentiality of genomic information that would be exceptional relative to the protections one would enact to protect other types of sensitive information. The special nature of genomic information lies in understanding that it is neither personal property nor mere information. A genome is, at one and the same time, a physical aspect of a person and information about that person. Genomic data are embodied information that partially constitutes as well as describes individuals and that connects them in physical ways to their ancestors and their relatives. All forms of privacy need to be protected, but some intimate aspects of our lives command special respect. To see a genome is more analogous to seeing a naked body than to seeing a social security number. This metaphor suggests that clinicians and investigators ought to respect the special concerns of patients regarding genomic information while not claiming that there are any exceptional measures one could take to protect genomic privacy. Suggestions are given for how this view might affect patient interactions, consent discussions, public policy, and public trust in genomic research and clinical genetics.

  17. Evolution of Circulating Wild Poliovirus and of Vaccine-Derived Poliovirus in an Immunodeficient Patient: a Unifying Model

    PubMed Central

    Gavrilin, Gene V.; Cherkasova, Elena A.; Lipskaya, Galina Y.; Kew, Olen M.; Agol, Vadim I.

    2000-01-01

    We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis. PMID:10906191

  18. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    PubMed

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

  19. [The vaccines based on the replicon of the venezuelan equine encephalomyelitis virus against viral hemorrhagic fevers].

    PubMed

    Petrov, A A; Plekhanova, T M; Sidorova, O N; Borisevich, S V; Makhlay, A A

    2015-01-01

    The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.

  20. Exploring Numerical (Naked) Singularity Formation with Collisionless Matter

    NASA Astrophysics Data System (ADS)

    Okounkova, Maria; Hemberger, Daniel; Scheel, Mark

    2017-01-01

    A proposed channel for the formation of naked singularities (singularities without event horizons) is the collapse of collisionless matter. In 1991, Shapiro and Teukolsky numerically investigated the collapse of prolate spheroids of collisionless matter in axisymmetry, and found that for certain initial configurations, a singularity formed on the domain without the appearance of an apparent horizon. While this may be a candidate for naked singularity formation, the role of the axisymmetry of the configuration and the termination of the simulation at singularity formation leave the question of generically forming an event horizon open. We have implemented (fully backreacting, fully 3-dimensional) collisionless matter evolution in SpEC, the Spectral Einstein Code, and present our results for the collapse of various configurations of collisionless matter. We expand on previous results by excising singularities, giving more time for the appearance of an apparent horizon, and by considering a variety of initial configurations.

  1. Conditions for the naked singularity formation in generalized Vaidya spacetime

    NASA Astrophysics Data System (ADS)

    Vertogradov, V. D.

    2016-11-01

    The gravitational collapse of generalised Vaidya spacetime is considered. It is known that the endstate of gravitational collapse, as to whether a black hole or a naked singularity is formed, depends on the mass function M(v,r). Here we give conditions for the mass function which corresponds to the equation of the state P = αρ where α ∈ (0, ⅓] and according to these conditions we obtain either a black hole or a naked singularity at the endstate of gravitational collapse. Also we give conditions for the mass function when the singularity is gravitationally strong. Also we provide the metric which is the analogue of Vaidya metric in case of rotation.

  2. Quantum particle probe of the Kerr naked singularity

    NASA Astrophysics Data System (ADS)

    Gurtug, O.; Halilsoy, M.

    2017-01-01

    We investigate Kerr's timelike naked singularity within the framework of quantum mechanics. A quantum particle in the form of a massive boson is sent in the plane θ = π/2 to the naked ring singularity of Kerr which develops for the overspinning case (a>M) to test it from a quantum picture. This singularity is analysed in two different coordinate systems. We show that the spatial operator of the Klein-Gordon equation both in Boyer-Lindquist and in the dragging coordinate systems has a unique self-adjoint extension. As a result, the classical Kerr's ring singularity becomes quantum regular, if it is probed with massive bosonic particles obeying the Klein-Gordon equation.

  3. Oral and Inactivated Poliovirus Vaccines in the Newborn: A review

    PubMed Central

    Mateen, Farrah J.; Shinohara, Russell T.; Sutter, Roland W.

    2015-01-01

    Background Oral poliovirus vaccine (OPV) remains the vaccine-of-choice for routine immunization and supplemental immunization activities (SIAs) to eradicate poliomyelitis globally. Recent data from India suggested lowerthanexpected immunogenicity of an OPV birth dose, prompting a review of the immunogenicity of OPV or inactivated poliovirus vaccine (IPV) when administered at birth. Methods We evaluated the seroconversion and reported adverse events among infants given a single birth dose (given ≤7 days of life) of OPV or IPV through a systematic review of published articles and conference abstracts from 1959-2011 in any language found on PubMed, Google Scholar, or reference lists of selected articles. Results 25 articles from 13 countries published between1959 and 2011 documented seroconversion rates in newborns following an OPV dose given within the first seven days of life. There were 10 studies that measured seroconversion rates between 4 and 8 weeks of a single birth dose of TOPV, using an umbilical cord blood draw at the time of birth to establish baseline antibody levels. The percentage of newborns who seroconverted at 8 weeks range 6-42% for poliovirus type 1, 2-63% for type 2, and 1-35% for type 3). For mOPV type 1, seroconversion ranged from 10-76%; mOPV type 3, the range was 12-58%; and for the one study reporting bOPV, it was 20% for type 1 and 7% for type 3. There were four studies of IPV in newborns with a seroconversion rate of 8-100% for serotype 1, 15-100% for serotype 2, and 15-94% for serotype 3, measured at 4-6 weeks of life. No serious adverse events related to newborn OPV or IPV dosing were reported, including no cases of acute flaccid paralysis. Conclusions There is great variability of the immunogenicity of a birth dose of OPV for reasons largely unknown. Our review confirms the utility of a birth dose of OPV, particularly in countries where early induction of polio immunity is imperative. IPV has higher seroconversion rates in newborns and

  4. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    PubMed Central

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  5. Poliovirus immunity in newly resettled adult refugees in Idaho, United States of America.

    PubMed

    Roscoe, Clay; Gilles, Ryan; Reed, Alex J; Messerschmidt, Matt; Kinney, Rebecca

    2015-06-12

    In the United States, vaccines have eliminated wild poliovirus (WPV) infection, though resettling refugees may lack immunity and importation of WPV remains a concern. A cross-sectional survey was performed to determine the prevalence of poliovirus immunity in adult refugees resettling in Boise, Idaho, U.S.A.; immunity was evaluated using two definitions: serotypes 1, 2 and 3 positive, or serotypes 1 and 3 positive. This survey evaluated 795 adult refugees between August 2010 and November 2012. Poliovirus immunity in adults >18 years was 55.3% for serotypes 1, 2 and 3 combined, and 60% for serotypes 1 and 3 only. This study demonstrated a WPV immunity rate of <60% in a recently resettled adult refugee population in the United States, reinforcing the need to ensure poliovirus immunity in all newly arrived adult refugees, either by expanding pre-departure immunization or by screening for immunity at resettlement and vaccinating when indicated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Comparison between adsorption of poliovirus and rotavirus by aluminum hydroxide and activated sludge flocs.

    PubMed Central

    Farrah, S R; Goyal, S M; Gerba, C P; Conklin, R H; Smith, E M

    1978-01-01

    Adsorption of poliovirus and rotavirus by aluminum hydroxide and activated sludge flocs was studied. Both aluminum hydroxide and activated sludge flocs adsorbed greater amounts of poliovirus than rotavirus. Aluminum hydroxide flocs reduced the titer of poliovirus in tap water by 3 log10, but they only reduced the titer of a simian rotovirus (SA-11) in tap water by 1 log10 or less and did not noticeably reduce the number of human rotavirus particles present in a dilute stool suspension. Activated sludge flocs reduced the titer of added poliovirus by 0.7 to 1.8 log10 and reduced the titer of SA-11 by 0.5 log10 or less. These studies indicate that a basic difference in the adsorptive behavior of enteroviruses and rotaviruses exists and that water and wastewater treatment processes that are highly effective in removal of enteroviruses may not be as effective in removing other viral groups such as rotaviruses. PMID:205173

  7. Why do naked singularities form in gravitational collapse? II

    SciTech Connect

    Joshi, Pankaj S.; Goswami, Rituparno; Dadhich, Naresh

    2004-10-15

    We examine physical features that could lead to formation of a naked singularity rather than black hole, as end state of spherical collapse. Generalizing earlier results on dust collapse to general type I matter fields, it is shown that collapse always creates black hole if shear vanishes or density is homogeneous. It follows that nonzero shear is a necessary condition for singularity to be visible to external observers, when trapped surface formation is delayed by shearing forces or inhomogeneity within the collapsing cloud.

  8. Naked-eye acquisition of visible near-earth satellites

    NASA Technical Reports Server (NTRS)

    Mansfield, Roger L.

    1988-01-01

    This paper develops visual sighting criteria for observing artificial near-earth satellites with the naked eye, and summarizes the mathematics needed to predict visibility efficiently from a set of mean orbital elements. It reports on a successful application of the sighting criteria and visibility-prediction mathematics, and shows that successful visual acquisition depends as much upon the observer's skill in recognizing the constellations as it does upon accurate visibility predictions.

  9. Naked-eye acquisition of visible near-earth satellites

    NASA Technical Reports Server (NTRS)

    Mansfield, Roger L.

    1988-01-01

    This paper develops visual sighting criteria for observing artificial near-earth satellites with the naked eye, and summarizes the mathematics needed to predict visibility efficiently from a set of mean orbital elements. It reports on a successful application of the sighting criteria and visibility-prediction mathematics, and shows that successful visual acquisition depends as much upon the observer's skill in recognizing the constellations as it does upon accurate visibility predictions.

  10. A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus.

    PubMed

    Korotkova, Ekaterina A; Gmyl, Anatoly P; Yakovenko, Maria L; Ivanova, Olga E; Eremeeva, Tatyana P; Kozlovskaya, Liubov I; Shakaryan, Armen K; Lipskaya, Galina Y; Parshina, Irina L; Loginovskikh, Nataliya V; Morozova, Nadezhda S; Agol, Vadim I

    2016-07-01

    Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly

  11. A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus

    PubMed Central

    Korotkova, Ekaterina A.; Gmyl, Anatoly P.; Yakovenko, Maria L.; Ivanova, Olga E.; Eremeeva, Tatyana P.; Kozlovskaya, Liubov I.; Shakaryan, Armen K.; Lipskaya, Galina Y.; Parshina, Irina L.; Loginovskikh, Nataliya V.; Morozova, Nadezhda S.

    2016-01-01

    ABSTRACT Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. IMPORTANCE The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused

  12. The Alpha/Beta Interferon Response Controls Tissue Tropism and Pathogenicity of Poliovirus

    PubMed Central

    Ida-Hosonuma, Miki; Iwasaki, Takuya; Yoshikawa, Tomoki; Nagata, Noriyo; Sato, Yuko; Sata, Tetsutaro; Yoneyama, Mitsutoshi; Fujita, Takashi; Taya, Choji; Yonekawa, Hiromichi; Koike, Satoshi

    2005-01-01

    Poliovirus selectively replicates in neurons in the spinal cord and brainstem, although poliovirus receptor (PVR) expression is observed in both the target and nontarget tissues in humans and transgenic mice expressing human PVR (PVR-transgenic mice). We assessed the role of alpha/beta interferon (IFN) in determining tissue tropism by comparing the pathogenesis of the virulent Mahoney strain in PVR-transgenic mice and PVR-transgenic mice deficient in the alpha/beta IFN receptor gene (PVR-transgenic/Ifnar knockout mice). PVR-transgenic/Ifnar knockout mice showed increased susceptibility to poliovirus. After intravenous inoculation, severe lesions positive for the poliovirus antigen were detected in the liver, spleen, and pancreas in addition to the central nervous system. These results suggest that the alpha/beta IFN system plays an important role in determining tissue tropism by protecting nontarget tissues that are potentially susceptible to infection. We subsequently examined the expression of IFN and IFN-stimulated genes (ISGs) in the PVR-transgenic mice. In the nontarget tissues, ISGs were expressed even in the noninfected state, and the expression level increased soon after poliovirus infection. On the contrary, in the target tissues, ISG expression was low in the noninfected state and sufficient response after poliovirus infection was not observed. The results suggest that the unequal IFN response is one of the important determinants for the differential susceptibility of tissues to poliovirus. We consider that poliovirus replication was observed in the nontarget tissues of PVR-transgenic/Ifnar knockout mice because the IFN response was null in all tissues. PMID:15767446

  13. A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

    PubMed Central

    Shepley, M P; Racaniello, V R

    1994-01-01

    A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways. Images PMID:7508992

  14. Poliovirus Studies during the Endgame of the Polio Eradication Program.

    PubMed

    Arita, Minetaro

    2017-01-24

    Since the beginning of Global Polio Eradication Initiative in 1988, poliomyelitis cases caused by wild poliovirus (PV) have been drastically reduced, with only 74 cases reported in 2 endemic countries in 2015. The current limited PV transmission suggests that we are in the endgame of the polio eradication program. However, specific challenges have emerged in the endgame, including tight budget, switching of the vaccines, and changes in biorisk management of PV. To overcome these challenges, several PV studies have been implemented in the eradication program. Some of the responses to the emerging challenges in the polio endgame might be valuable in other infectious diseases eradication programs. Here, I will review challenges that confront the polio eradication program and current research to address these challenges.

  15. The Sabin live poliovirus vaccination trials in the USSR, 1959.

    PubMed Central

    Horstmann, D. M.

    1991-01-01

    Widespread use of the Sabin live attenuated poliovirus vaccine has had tremendous impact on the disease worldwide, virtually eliminating it from a number of countries, including the United States. Early proof of its safety and effectiveness was presented in 1959 by Russian investigators, who had staged massive trials in the USSR, involving millions of children. Their positive results were at first viewed in the United States and elsewhere with some skepticism, but the World Health Organization favored proceeding with large-scale trials, and responded to the claims made by Russian scientists by sending a representative to the USSR to review in detail the design and execution of the vaccine programs and the reliability of their results. The report that followed was a positive endorsement of the findings and contributed to the acceptance of the Sabin vaccine in the United States, where it has been the polio vaccine of choice since the mid-1960s. PMID:1814062

  16. Parallel path: poliovirus research in the vaccine era.

    PubMed

    Garfinkel, Michele S; Sarewitz, Daniel

    2003-07-01

    One goal of the scientific research enterprise is to improve the lives of individuals and the overall health of societies. This goal is achieved through a combination of factors, including the composition of research portfolios. In turn, this composition is determined by a variety of scientific and societal needs. The recent history of polio research highlights the complex relations between research policy, scientific progress and societal benefits. Here, we briefly review the circumstances leading to the possibility of eradication of poliovirus, evaluate the research environment that emerged following the introduction of a vaccine, and compare and contrast the current research framework with that for other infectious diseases. From this analysis, policy lessons with general applicability to scientific research are identified.

  17. Can post-eradication laboratory containment of wild polioviruses be achieved?

    PubMed Central

    Dowdle, Walter R.; Gary, Howard E.; Sanders, Raymond; van Loon, Anton M.

    2002-01-01

    The purpose of containment is to prevent reintroduction of wild polioviruses from laboratories into polio-free communities. In order to achieve global commitment to laboratory containment the rationale should be clear and compelling; the biosafety levels should be justified by the risks; and the objectives should be realistic. Absolute containment can never be assured. Questions of intentional or unintentional non-compliance can never be wholly eliminated. Effective laboratory containment is, however, a realistic goal. Prevention of virus transmission through contaminated laboratory materials is addressed by WHO standards for biosafety. The principal challenge is to prevent transmission through unrecognized infectious laboratory workers. Such transmission is possible only if the following conditions occur: infectious and potentially infectious materials carrying wild poliovirus are present in the laboratory concerned; a laboratory operation exposes a worker to poliovirus; a worker is susceptible to an infection that results in the shedding of poliovirus; and the community is susceptible to poliovirus infections. At present it is difficult to envisage the elimination of any of these conditions. However, the risks of the first three can be greatly reduced so as to create a formidable barrier against poliovirus transmission to the community. Final biosafety recommendations must await post-eradication immunization policies adopted by the international community. PMID:12075368

  18. The Multifaceted Poliovirus 2A Protease: Regulation of Gene Expression by Picornavirus Proteases

    PubMed Central

    Castelló, Alfredo; Álvarez, Enrique; Carrasco, Luis

    2011-01-01

    After entry into animal cells, most viruses hijack essential components involved in gene expression. This is the case of poliovirus, which abrogates cellular translation soon after virus internalization. Abrogation is achieved by cleavage of both eIF4GI and eIF4GII by the viral protease 2A. Apart from the interference of poliovirus with cellular protein synthesis, other gene expression steps such as RNA and protein trafficking between nucleus and cytoplasm are also altered. Poliovirus 2Apro is capable of hydrolyzing components of the nuclear pore, thus preventing an efficient antiviral response by the host cell. Here, we compare in detail poliovirus 2Apro with other viral proteins (from picornaviruses and unrelated families) as regard to their activity on key host factors that control gene expression. It is possible that future analyses to determine the cellular proteins targeted by 2Apro will uncover other cellular functions ablated by poliovirus infection. Further understanding of the cellular proteins hydrolyzed by 2Apro will add further insight into the molecular mechanism by which poliovirus and other viruses interact with the host cell. PMID:21541224

  19. Wild and vaccine-derived poliovirus circulation, and implications for polio eradication.

    PubMed

    Lopalco, P L

    2017-02-01

    Polio cases due to wild virus are reported by only three countries in the world. Poliovirus type 2 has been globally eradicated and the last detection of poliovirus type 3 dates to November 2012. Poliovirus type 1 remains the only circulating wild strain; between January and September 2016 it caused 26 cases (nine in Afghanistan, 14 in Pakistan, three in Nigeria). The use of oral polio vaccine (OPV) has been the key to success in the eradication effort. However, paradoxically, moving towards global polio eradication, the burden caused by vaccine-derived polioviruses (VDPVs) becomes increasingly important. In this paper circulation of both wild virus and VDPVs is reviewed and implications for the polio eradication endgame are discussed. Between April and May 2016 OPV2 cessation has been implemented globally, in a coordinated switch from trivalent OPV to bivalent OPV. In order to decrease the risk for cVDPV2 re-emergence inactivated polio vaccine (IPV) has been introduced in the routine vaccine schedule of all countries. The likelihood of re-emergence of cVDPVs should markedly decrease with time after OPV cessation, but silent circulation of polioviruses cannot be ruled out even a long time after cessation. For this reason, immunity levels against polioviruses should be kept as high as possible in the population by the use of IPV, and both clinical and environmental surveillance should be maintained at a high level.

  20. Contribution of Environmental Surveillance Toward Interruption of Poliovirus Transmission in Nigeria, 2012–2015

    PubMed Central

    Johnson Muluh, Ticha; Hamisu, Abdullahi Walla; Craig, Kehinde; Mkanda, Pascal; Andrew, Etsano; Adeniji, Johnson; Akande, Adefunke; Musa, Audu; Ayodeji, Isiaka; Nicksy, Gumede; Banda, Richard; Tegegne, Sisay G.; Nsubuga, Peter; Oyetunji, Ajiboye; Diop, Ousmane; Vaz, Rui G.; Muhammad, Ado J. G.

    2016-01-01

    Background. Cases of paralysis caused by poliovirus have decreased by >99% since the 1988 World Health Assembly's resolution to eradicate polio. The World Health Organization identified environmental surveillance (ES) of poliovirus in the poliomyelitis eradication strategic plan as an activity that can complement acute flaccid paralysis (AFP) surveillance. This article summarizes key public health interventions that followed the isolation of polioviruses from ES between 2012 and 2015. Methods. The grap method was used to collect 1.75 L of raw flowing sewage every 2–4 weeks. Once collected, samples were shipped at 4°C to a polio laboratory for concentration. ES data were then used to guide program implementation. Results. From 2012 to 2015, ES reported 97 circulating vaccine-derived polioviruses (cVDPV2) and 14 wild polioviruses. In 2014 alone, 54 cVDPV type 2 cases and 1 WPV type 1 case were reported. In Sokoto State, 58 cases of AFP were found from a search of 9426 households. A total of 2 252 059 inactivated polio vaccine and 2 460 124 oral polio vaccine doses were administered to children aged <5 year in Borno and Yobe states. Conclusions. This article is among the first from Africa that relates ES findings to key public health interventions (mass immunization campaigns, inactivated polio vaccine introduction, and strengthening of AFP surveillance) that have contributed to the interruption of poliovirus transmission in Nigeria. PMID:26908747

  1. Identification of vaccine-related polioviruses by hybridization with specific RNA probes.

    PubMed Central

    De, L; Nottay, B; Yang, C F; Holloway, B P; Pallansch, M; Kew, O

    1995-01-01

    We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5'-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5'-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamant ane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes. PMID:7751358

  2. Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria.

    PubMed

    Korsun, Neli; Kojouharova, Mira; Vladimirova, Nadezhda; Fiore, Lucia; Litvinenko, Ivan; Buttinelli, Gabriele; Fiore, Stefano; Voynova-Georgieva, Violeta; Mladenova, Zornitsa; Georgieva, Daniela

    2009-09-01

    Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine-associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine-associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4-month-old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin-like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U-to-C back mutation at nt 472 of the 5' UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine-associated paralytic poliomyelitis in all the three patients.

  3. Poliovirus and other enteroviruses in children infected with intestinal parasites in Nigeria.

    PubMed

    Adekolujo, Daniel R; Olayinka, Suraj O; Adeniji, Johnson A; Oyeyemi, Oyetunde T; Odaibo, Alexander B

    2015-10-29

    Poliovirus, an enterovirus, still persists in Nigeria despite the global efforts tailored towards its eradication. This study aimed to assess the impacts of poliovirus and other enteroviruses on the susceptibility of individuals to intestinal parasite infections. A cross-sectional study on the prevalence of intestinal parasites was conducted on two-sample stool specimens of 717 Nigerian children (between 1 and 19 years of age) whose poliovirus/other enteroviruses infection status had been determined. The overall prevalence of Sabin poliovirus and other related enteroviruses infections were 6.6% and 13.8%, respectively. The prevalence of Ascaris lumbricoides was significantly higher than that of other intestinal parasites (p < 0.05), with children in the 0-4 year age group being the most predisposed age group to intestinal parasitic infection (OR = 11.7, CI = 9.2-15.0). While the prevalence of all species of parasites except S. mansoni showed no significant variations in children with Sabin poliovirus (p > 0.05), the prevalence of hookworms and Taenia spp. was significantly higher in children with other enteroviral infections (p < 0.05). The high risk of children of acquiring enteroviral infection through some intestinal parasites is an indication of possible association of the parasites in a more poliovirus-endemic population. A combined intervention approach for the two infections is advocated.

  4. Framework for evaluating the risks of paralytic poliomyelitis after global interruption of wild poliovirus transmission.

    PubMed Central

    Aylward, R. Bruce; Cochi, Stephen L.

    2004-01-01

    With the interruption of wild poliovirus transmission globally, the need for new policies to deal with the post-certification era will rapidly arise. New policies will be required in four areas: detection and notification of circulating polioviruses; biocontainment of wild, vaccine-derived and attenuated strains of poliovirus; vaccine stockpiles and response mechanisms; and routine immunization against polioviruses. A common understanding of the potential risks of paralytic poliomyelitis in the post-certification period is essential to the development of these policies. Since 2000, there has been increasing international consensus that the risks of paralytic poliomyelitis in the post-certification era fall into two categories: those due to the continued use of the oral poliovirus vaccine (OPV) and those due to future improper handling of wild polioviruses. The specific risks within both categories have now been defined, and an understanding of the frequency and potential burden of disease associated with each is rapidly improving. This knowledge and clarity have provided a framework that is already proving valuable for identifying research priorities and discussing potential policy options with national authorities. However, this framework must be regarded as a dynamic tool, requiring regular updating as additional information on these risks becomes available through further scientific research, programmatic work, and policy decisions. PMID:15106299

  5. Prolonged Replication of a Type 1 Vaccine-Derived Poliovirus in an Immunodeficient Patient

    PubMed Central

    Kew, Olen M.; Sutter, Roland W.; Nottay, Baldev K.; McDonough, Michael J.; Prevots, D. Rebecca; Quick, Linda; Pallansch, Mark A.

    1998-01-01

    VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by ∼10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of ∼3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started ∼9.3 years earlier. This estimate is about the time (6.9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication. PMID:9738040

  6. Immunization of newborn children with living oral trivalent poliovirus vaccine.

    PubMed

    CAMPILLO-SAINZ, C; ORNELAS HERNANDEZ, A; DE MUCHA MACIAS, J; NAVA, S E

    1962-09-01

    Campillo-Sainz, C. (Instituto Nacional de Virología de la S.S.A., México, D.F.), A. Ornelas Hernandez, J. de Mucha Macías, and S. E. Nava. Immunization of newborn children with living oral trivalent poliovirus vaccine. J. Bacteriol. 84:446-450. 1962.-The serological response to one dose of living oral trivalent polio-virus vaccine was compared in two groups of children, 49 vaccinated at birth and 44 vaccinated at the age of 4 months. Of those vaccinated at birth, 44 (90%) responded to the vaccine strains of type 1 and type 3 and 30 (61%) to the type 2 strain. Of those vaccinated at 4 months of age; 64% responded to type 1, 52% to type 2, and 82% to type 3. The difference between the responses of the two groups, which for type 1 is significant, may result from the interference of other enteric viruses in the 4-month-old children. A second dose of vaccine, administered to the children vaccinated at birth when they reached the age of 4 months, increased the over-all immunological response to 100% for types 1 and 3 and 96% for type 2, and showed that no immunological tolerance had been developed. The vaccine produced no undesirable effects in any of the children, and no paralytic poliomyelitis occurred among them. The observation of other investigators, that a high titer of maternal antibody inhibits immunological response to vaccination, was confirmed, but breast feeding apparently had no unfavorable effect on response.

  7. THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY

    PubMed Central

    Svehag, Sven-Eric; Mandel, Benjamin

    1964-01-01

    Rapid formation of poliovirus-neutralizing antibody was observed in the rabbit. 19S type antibody was detectable 8 to 12 hours following a single intravenous virus injection and the induction period was of the order of 4 to 5 hours or less. The production of 7S antibody had a longer lag phase (1½ to 2 days) and it was formed at slower rate. The observed rate of early 19S and 7S antibody formation as well as the peak titers of the two antibodies were antigen dose dependent. Normal rabbit sera showed low neutralizing activity to several viral antigens in a sensitive assay system. Following intravenous inoculation of poliovirus either transitory (⩽ 1 month) or enduring (¾ to 1½ year) antibody formation resulted depending upon the dose of antigen employed. In transitory responses, which could be induced by a single small antigen dose, only 19S antibody was demonstrable and there was an abrupt cessation of antibody synthesis on day 4 or 5. In enduring responses, both 19S and 7S antibody were formed and the minimum antigen dose required for initiation of such a response was equal to the dose needed for induction of 7S antibody formation. Thus, enduring antibody formation was an all-or-none phenomenon depending upon whether or not 7S antibody formation was induced. The antigen dose requirement for induction of 7S antibody was much higher (by 50-fold or more) than that for 19S antibody. This allowed a determination of antigen dose regions, within which predictably transitory (19S) or enduring (19S + 7S) antibody formation was obtained. These pronounced differences in antigen dose requirement for induction and kinetics of formation of 19S and 7S antibody suggest that the same cells do not participate in the formation of the two antibodies. PMID:14113113

  8. IMMUNIZATION OF NEWBORN CHILDREN WITH LIVING ORAL TRIVALENT POLIOVIRUS VACCINE

    PubMed Central

    Campillo-Sainz, C.; Hernandez, A. Ornelas; MacÍas, J. de Mucha; Nava, S. E.

    1962-01-01

    Campillo-Sainz, C. (Instituto Nacional de Virología de la S.S.A., México, D.F.), A. Ornelas Hernandez, J. de Mucha Macías, and S. E. Nava. Immunization of newborn children with living oral trivalent poliovirus vaccine. J. Bacteriol. 84:446–450. 1962.—The serological response to one dose of living oral trivalent polio-virus vaccine was compared in two groups of children, 49 vaccinated at birth and 44 vaccinated at the age of 4 months. Of those vaccinated at birth, 44 (90%) responded to the vaccine strains of type 1 and type 3 and 30 (61%) to the type 2 strain. Of those vaccinated at 4 months of age; 64% responded to type 1, 52% to type 2, and 82% to type 3. The difference between the responses of the two groups, which for type 1 is significant, may result from the interference of other enteric viruses in the 4-month-old children. A second dose of vaccine, administered to the children vaccinated at birth when they reached the age of 4 months, increased the over-all immunological response to 100% for types 1 and 3 and 96% for type 2, and showed that no immunological tolerance had been developed. The vaccine produced no undesirable effects in any of the children, and no paralytic poliomyelitis occurred among them. The observation of other investigators, that a high titer of maternal antibody inhibits immunological response to vaccination, was confirmed, but breast feeding apparently had no unfavorable effect on response. PMID:14018173

  9. Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

    PubMed

    Sanders, Barbara P; Liu, Ying; Brandjes, Alies; van Hoek, Vladimir; de Los Rios Oakes, Isabel; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2015-09-01

    Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

  10. Myristoylation is important at multiple stages in poliovirus assembly.

    PubMed Central

    Moscufo, N; Simons, J; Chow, M

    1991-01-01

    The N-terminal glycine of the VP4 capsid subunit of poliovirus is covalently modified with myristic acid (C14 saturated fatty acid). To investigate the function of VP4 myristoylation in poliovirus replication, amino acid substitutions were placed within the myristoylation consensus sequence at the alanine residue (4003A) adjacent to the N-terminal glycine by using site-directed mutagenesis methods. Mutants which replace the alanine residue with a small hydrophobic residue such as leucine, valine, or glycine displayed normal levels of myristoylation and normal growth kinetics. Replacement with the polar amino acid histidine (4003A.H) also resulted in a level of myristoylation comparable to that of the wild type. However, replacement of the alanine residue with aspartic acid (4003A.D) caused a dramatic reduction (about 40 to 60%) in myristoylation levels of the VP4 precursors (P1 and VP0). In contrast, no differences in modification levels were found in either VP0 and VP4 proteins isolated from mature mutant virions, indicating that myristoylation is required for assembly of the infectious virion. The myristoylation levels of the VP0 proteins found in capsid assembly intermediates indicate that there is a strong but not absolute preference for myristoyl-modified subunits during pentamer formation. Complete myristoylation was observed in mature virions but not in assembly intermediates, indicating that there is a selection for myristoyl-modified subunits during stable RNA encapsidation to form the mature virus particle. In addition, even though mutant infectious virions are fully modified, the severe reduction in specific infectivity of both 4003A.D and 4003A.H purified viruses indicates that the amino acid residue adjacent to the N-terminal glycine apparently has an additional role early during viral infection and that mutations at this position induce pleiotropic effects. Images PMID:1850017

  11. Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

    PubMed Central

    Barton, David J.; Morasco, B. Joan; Flanegan, James B.

    1999-01-01

    Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated

  12. Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

    PubMed

    Manzano, Mark; Padmanabhan, Radhakrishnan

    2014-01-01

    The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

  13. Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

    PubMed Central

    Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M.; Rossolini, Gian Maria

    2006-01-01

    Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  14. Replicon typing of plasmids encoding resistance to newer beta-lactams.

    PubMed

    Carattoli, Alessandra; Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M; Rossolini, Gian Maria

    2006-07-01

    Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

  15. Enhanced potency of replicon vaccine using one vector to simultaneously co-express antigen and interleukin-4 molecular adjuvant

    PubMed Central

    Ma, Yao; An, Huai-Jie; Wei, Xiao-Qi; Xu, Qing; Yu, Yun-Zhou; Sun, Zhi-Wei

    2013-01-01

    We evaluated the utility of interleukin-4 (IL-4) as molecular adjuvant of replicon vaccines for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In both Balb/c and C57/BL6 mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) encoding the Hc gene of BoNT/A (AHc), the immunogenicity was significantly modulated and enhanced by co-delivery or co-express of the IL-4 molecular adjuvant. The enhanced potencies were also produced by co-delivery or co-expression of the IL-4 molecular adjuvant in mice immunized with the recombinant SFV replicon particles (VRP) vaccines. In particular, when AHc and IL-4 were co-expressed within the same replicon vaccine vector using dual-expression or bicistronic IRES, the anti-AHc antibody titers, serum neutralization titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased. These results indicate IL-4 is an effective Th2-type adjuvant for the replicon vaccines in both strain mice, and the co-expression replicon vaccines described here may be an excellent candidate for further vaccine development in other animals or humans. Thus, we described a strategy to design and develop efficient vaccines against BoNT/A or other pathogens using one replicon vector to simultaneously co-express antigen and molecular adjuvant. PMID:23291932

  16. Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

    PubMed Central

    Akhtar, Parvez; Khan, Saleem A.

    2014-01-01

    The large pXO1 plasmid (181.6 kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid. PMID:22239982

  17. Poliovirus excretion among persons with primary immune deficiency disorders: summary of a seven-country study series.

    PubMed

    Li, Li; Ivanova, Olga; Driss, Nadia; Tiongco-Recto, Marysia; da Silva, Rajiva; Shahmahmoodi, Shohreh; Sazzad, Hossain M S; Mach, Ondrej; Kahn, Anna-Lea; Sutter, Roland W

    2014-11-01

    Persons with primary immune deficiency disorders (PID), especially those disorders affecting the B-cell system, are at substantially increased risk of paralytic poliomyelitis and can excrete poliovirus chronically. However, the risk of prolonged or chronic excretion is not well characterized in developing countries. We present a summary of a country study series on poliovirus excretion among PID cases. Cases with PID from participating institutions were enrolled during the first year and after obtaining informed consent were tested for polioviruses in stool samples. Those cases excreting poliovirus were followed on a monthly basis during the second year until 2 negative stool samples were obtained. A total of 562 cases were enrolled in Bangladesh, China, Iran, Philippines, Russia, Sri Lanka, and Tunisia during 2008-2013. Of these, 17 (3%) shed poliovirus, including 2 cases with immunodeficient vaccine-derived poliovirus. Poliovirus was detected in a single sample from 5/17 (29%) cases. One case excreted for more than 6 months. None of the cases developed paralysis during the study period. Chronic polioviruses excretion remains a rare event even among individuals with PID. Nevertheless, because these individuals were not paralyzed they would have been missed by current surveillance; therefore, surveillance for polioviruses among PID should be established. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

    PubMed Central

    Boylan, Brendan T.; Moreira, Fernando R.; Carlson, Tim W.

    2017-01-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses. PMID:28187142

  19. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

    PubMed

    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  20. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    PubMed

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-05-01

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

    PubMed Central

    Heinrich, J; Riedel, H D; Rückert, B; Lurz, R; Schuster, H

    1995-01-01

    The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon. Images PMID:7784198

  2. An evaluation of the sensitivity of acute flaccid paralysis surveillance for poliovirus infection in Australia

    PubMed Central

    2009-01-01

    Background World Health Organization (WHO) targets for acute flaccid paralysis (AFP) surveillance, including the notification of a minimum rate of AFP among children, are used to assess the adequacy of AFP surveillance for the detection of poliovirus infection. Sensitive surveillance for poliovirus infection in both developed and developing countries is essential to support global disease eradication efforts. We applied recently developed methods for the quantitative evaluation of disease surveillance systems to evaluate the sensitivity of AFP surveillance for poliovirus infection in Australia. Methods A scenario tree model which accounted for administrative region, age, population immunity, the likelihood of AFP, and the probability of notification and stool sampling was used to assess the sensitivity of AFP surveillance for wild poliovirus infection among children aged less than 15 years in Australia. The analysis was based on historical surveillance data collected between 2000 and 2005. We used a surveillance time period of one month, and evaluated the ability of the surveillance system to detect poliovirus infection at a prevalence of 1 case per 100 000 persons and 1 case per million persons. Results There was considerable variation in the sensitivity of AFP surveillance for poliovirus infection among Australian States and Territories. The estimated median sensitivity of AFP surveillance in Australia among children aged less than 15 years was 8.2% per month at a prevalence of 1 case per 100,000 population, and 0.9% per month at a prevalence of 1 case per million population. The probability that Australia is free from poliovirus infection given negative surveillance findings following 5 years of continuous surveillance was 96.9% at a prevalence of 1 case per 100,000 persons and 56.5% at a prevalence of 1 case per million persons. Conclusion Given the ongoing risk of poliovirus importation prior to global eradication, long term surveillance is required to provide

  3. Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

    SciTech Connect

    Van't Hof, J.

    1980-01-01

    DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16 to 27 x 10/sup 6/D) and apparent cluster (approx. 330 x 10/sup 6/D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 x 10/sup 3/ clusters, and 7.7 x 10/sup 4/ replicons per genome.

  4. Inactivation of Poliovirus Type 1 by the Kelly-Purdy Ultraviolet Seawater Treatment Unit1

    PubMed Central

    Hill, William F.; Hamblet, Frederick E.; Benton, William H.

    1969-01-01

    Three experiments were conducted to determine the effect of ultraviolet (UV) radiation on poliovirus-contaminated seawater. In two of the experiments, the effectiveness of the Kelly-Purdy UV Seawater Treatment Unit to inactivate poliovirus type 1 (T1) suspended in continuously flowing seawater was determined. In experiment 1, the observed survival ratio of poliovirus T1 was 2.3 × 10-4 (99.98% reduction) in 15.7 sec. No virus was detected (<0.2 plaque-forming unit/ml) in 20.6 seconds. The calculated half-life value was 1.29 sec. In experiment 2, the observed survival ratio of poliovirus T1 was 5.9 × 10-4 (99.94% reduction) in 11.7 sec. No virus was detected in 15.7 sec. The calculated half-life value was 1.37 sec. In experiment 3, a laboratory-controlled UV experiment designed to closely simulate the geometry of the continuously flowing seawater system, the observed survival ratios of poliovirus T1 were 9.7 × 10-3 (99.03% reduction) and 3.6 × 10-4 (99.96% reduction) in 15 and 30 sec, respectively; the calculated half-life value was 2.38 sec. A statistically significant difference was found between the inactivation rates of poliovirus T1 in the two test systems. This rate difference was attributed primarily to UV dosage and stirring effects. The data indicated that UV radiation effectively inactivated poliovirus T1 in flowing seawater. These results validate the efficacy of the Kelly-Purdy UV Seawater Treatment Unit for use in commercial depuration systems. Images PMID:4304572

  5. Inactivation of poliovirus type 1 by the Kelly-Purdy ultraviolet seawater treatment unit.

    PubMed

    Hill, W F; Hamblet, F E; Benton, W H

    1969-01-01

    Three experiments were conducted to determine the effect of ultraviolet (UV) radiation on poliovirus-contaminated seawater. In two of the experiments, the effectiveness of the Kelly-Purdy UV Seawater Treatment Unit to inactivate poliovirus type 1 (T(1)) suspended in continuously flowing seawater was determined. In experiment 1, the observed survival ratio of poliovirus T(1) was 2.3 x 10(-4) (99.98% reduction) in 15.7 sec. No virus was detected (<0.2 plaque-forming unit/ml) in 20.6 seconds. The calculated half-life value was 1.29 sec. In experiment 2, the observed survival ratio of poliovirus T(1) was 5.9 x 10(-4) (99.94% reduction) in 11.7 sec. No virus was detected in 15.7 sec. The calculated half-life value was 1.37 sec. In experiment 3, a laboratory-controlled UV experiment designed to closely simulate the geometry of the continuously flowing seawater system, the observed survival ratios of poliovirus T(1) were 9.7 x 10(-3) (99.03% reduction) and 3.6 x 10(-4) (99.96% reduction) in 15 and 30 sec, respectively; the calculated half-life value was 2.38 sec. A statistically significant difference was found between the inactivation rates of poliovirus T(1) in the two test systems. This rate difference was attributed primarily to UV dosage and stirring effects. The data indicated that UV radiation effectively inactivated poliovirus T(1) in flowing seawater. These results validate the efficacy of the Kelly-Purdy UV Seawater Treatment Unit for use in commercial depuration systems.

  6. World Health Organization Guidelines for Containment of Poliovirus Following Type-Specific Polio Eradication - Worldwide, 2015.

    PubMed

    Previsani, Nicoletta; Tangermann, Rudolph H; Tallis, Graham; Jafari, Hamid S

    2015-08-28

    In 1988, the World Health Assembly of the World Health Organization (WHO) resolved to eradicate polio worldwide. Among the three wild poliovirus (WPV) types (type 1, type 2, and type 3), WPV type 2 (WPV2) has been eliminated in the wild since 1999, and WPV type 3 (WPV3) has not been reported since 2012. In 2015, only Afghanistan and Pakistan have reported WPV transmission. On May 25, 2015, all WHO Member States endorsed World Health Assembly resolution 68.3 on full implementation of the Polio Eradication and Endgame Strategic Plan 2013-2018 (the Endgame Plan), and with it, the third Global Action Plan to minimize poliovirus facility-associated risk (GAPIII). All WHO Member States have committed to implementing appropriate containment of WPV2 in essential laboratory and vaccine production facilities* by the end of 2015 and of type 2 oral poliovirus vaccine (OPV2) within 3 months of global withdrawal of OPV2, which is planned for April 2016. This report summarizes critical steps for essential laboratory and vaccine production facilities that intend to retain materials confirmed to contain or potentially containing type-specific WPV, vaccine-derived poliovirus (VDPV), or OPV/Sabin viruses, and steps for nonessential facilities† that process specimens that contain or might contain polioviruses. National authorities will need to certify that the essential facilities they host meet the containment requirements described in GAPIII. After certification of WPV eradication, the use of all OPV will cease; final containment of all polioviruses after polio eradication and OPV cessation will minimize the risk for reintroduction of poliovirus into a polio-free world.

  7. Seroprevalence of poliovirus antibodies in the Kansas City metropolitan area, 2012-2013.

    PubMed

    Wallace, Gregory S; Pahud, Barbara A; Weldon, William C; Curns, Aaron T; Oberste, M Steven; Harrison, Christopher J

    2017-04-03

    No indigenous cases of poliomyelitis have occurred in the US since 1979; however the risk of importation persists until global eradication is achieved. The seropositivity rate for different age cohorts with exposures to different poliovirus vaccine types and wild virus in the US are not presently known. A convenience sample was conducted in the Kansas City metropolitan area during 2012-2103 with approximately 100 participants enrolled for each of 5 age cohorts categorized based on vaccine policy changes over time in the US. Immunization records for poliovirus vaccination were required for participants <18 y of age. We evaluated the prevalence of serum antibodies to all 3 poliovirus serotypes. Seroprevalence was evaluated by demographics as well as between polio serotypes. The overall seroprevalence to poliovirus was 90.7%, 94.4%, and 83.3%, for types 1, 2, and 3, respectively. Seroprevalence was high (88.6%-96.2%) for all 3 types of poliovirus for the 6-10 y old age group that was likely to have received a complete schedule of IPV-only vaccination. Children 2-3 y of age, who have not yet completed their full IPV series, had lower seroprevalence compared with all older age groups for types 1 and 2 (p-value <0. 05). Seroprevalence was high for all 3 types of poliovirus in the population surveyed. Seroprevalence for subjects aged 2-3 y was lower than all other age groups for serotypes 1 and 2 highlighting the importance of completing the recommended poliovirus vaccine series with a booster dose at age 4-6 y.

  8. Metamaterial optical refractive index sensor detected by the naked eye

    NASA Astrophysics Data System (ADS)

    Wang, Xiaonong; Luo, Chunrong; Hong, Gang; Zhao, Xiaopeng

    2013-03-01

    An optical refractive index sensor that can be detected by the naked eye is presented in this paper. The sensor is developed based on the theory that metamaterials can completely absorb incident electric and magnetic field near its resonant frequencies. By inserting different mediums in the sensor or by dropping the mediums on the surface of the sensor, we detect the medium with a different refractive index through qualitative and quantitative analysis. The sensor is convenient for the detection of the liquid refractive index.

  9. Is it Really Naked? On Cosmic Censorship in String Theory

    SciTech Connect

    Frolov, A

    2004-09-30

    We investigate the possibility of cosmic censorship violation in string theory using a characteristic double-null code, which penetrates horizons and is capable of resolving the spacetime all the way to the singularity. We perform high-resolution numerical simulations of the evolution of negative mass initial scalar field profiles, which were argued to provide a counter example to cosmic censorship conjecture for AdS-asymptotic spacetimes in five-dimensional supergravity. In no instances formation of naked singularity is seen. Instead, numerical evidence indicates that black holes form in the collapse. Our results are consistent with earlier numerical studies, and explicitly show where the ''no black hole'' argument breaks.

  10. Is it really naked? On cosmic censorship in string theory

    SciTech Connect

    Frolov, Andrei V.

    2004-11-15

    We investigate the possibility of cosmic censorship violation in string theory using a characteristic double-null code, which penetrates horizons and is capable of resolving the spacetime all the way to the singularity. We perform high-resolution numerical simulations of the evolution of negative mass initial scalar field profiles, which were argued to provide a counterexample to cosmic censorship conjecture for AdS-asymptotic spacetimes in five-dimensional supergravity. In no instances formation of naked singularity is seen. Instead, numerical evidence indicates that black holes form in the collapse. Our results are consistent with earlier numerical studies, and explicitly show where the 'no black hole' argument breaks.

  11. Back to basics: naked-eye astronomical observation

    NASA Astrophysics Data System (ADS)

    Barclay, Charles

    2003-09-01

    For pupils of both sexes and all ages from about six upwards, the subject of Astronomy holds many fascinations - the rapid changes in knowledge, the large resource of available IT packages and above all the beautiful pictures from Hubble and the large Earth-based telescopes. This article, however, stresses the excitement and importance of naked-eye (unaided) first-hand observation, where light pollution allows, and suggests some techniques that may be used to enthuse and introduce youngsters to the glory of the night sky without recourse to computer screens.

  12. Time delay and magnification centroid due to gravitational lensing by black holes and naked singularities

    SciTech Connect

    Virbhadra, K. S.; Keeton, C. R.

    2008-06-15

    We model the massive dark object at the center of the Galaxy as a Schwarzschild black hole as well as Janis-Newman-Winicour naked singularities, characterized by the mass and scalar charge parameters, and study gravitational lensing (particularly time delay, magnification centroid, and total magnification) by them. We find that the lensing features are qualitatively similar (though quantitatively different) for Schwarzschild black holes, weakly naked, and marginally strongly naked singularities. However, the lensing characteristics of strongly naked singularities are qualitatively very different from those due to Schwarzschild black holes. The images produced by Schwarzschild black hole lenses and weakly naked and marginally strongly naked singularity lenses always have positive time delays. On the other hand, strongly naked singularity lenses can give rise to images with positive, zero, or negative time delays. In particular, for a large angular source position the direct image (the outermost image on the same side as the source) due to strongly naked singularity lensing always has a negative time delay. We also found that the scalar field decreases the time delay and increases the total magnification of images; this result could have important implications for cosmology. As the Janis-Newman-Winicour metric also describes the exterior gravitational field of a scalar star, naked singularities as well as scalar star lenses, if these exist in nature, will serve as more efficient cosmic telescopes than regular gravitational lenses.

  13. Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

    PubMed

    Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

    2015-10-13

    Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents.

  14. Increasing Type 1 Poliovirus Capsid Stability by Thermal Selection

    PubMed Central

    Adeyemi, Oluwapelumi O.; Nicol, Clare

    2016-01-01

    ABSTRACT Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the “empty” capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines. IMPORTANCE Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not

  15. Preventing Vaccine-Derived Poliovirus Emergence during the Polio Endgame

    PubMed Central

    Burns, Cara C.; Lyons, Hil; Blake, Isobel M.; Oberste, M. Steven; Kew, Olen M.; Grassly, Nicholas C.

    2016-01-01

    Reversion and spread of vaccine-derived poliovirus (VDPV) to cause outbreaks of poliomyelitis is a rare outcome resulting from immunisation with the live-attenuated oral poliovirus vaccines (OPVs). Global withdrawal of all three OPV serotypes is therefore a key objective of the polio endgame strategic plan, starting with serotype 2 (OPV2) in April 2016. Supplementary immunisation activities (SIAs) with trivalent OPV (tOPV) in advance of this date could mitigate the risks of OPV2 withdrawal by increasing serotype-2 immunity, but may also create new serotype-2 VDPV (VDPV2). Here, we examine the risk factors for VDPV2 emergence and implications for the strategy of tOPV SIAs prior to OPV2 withdrawal. We first developed mathematical models of VDPV2 emergence and spread. We found that in settings with low routine immunisation coverage, the implementation of a single SIA increases the risk of VDPV2 emergence. If routine coverage is 20%, at least 3 SIAs are needed to bring that risk close to zero, and if SIA coverage is low or there are persistently “missed” groups, the risk remains high despite the implementation of multiple SIAs. We then analysed data from Nigeria on the 29 VDPV2 emergences that occurred during 2004−2014. Districts reporting the first case of poliomyelitis associated with a VDPV2 emergence were compared to districts with no VDPV2 emergence in the same 6-month period using conditional logistic regression. In agreement with the model results, the odds of VDPV2 emergence decreased with higher routine immunisation coverage (odds ratio 0.67 for a 10% absolute increase in coverage [95% confidence interval 0.55−0.82]). We also found that the probability of a VDPV2 emergence resulting in poliomyelitis in >1 child was significantly higher in districts with low serotype-2 population immunity. Our results support a strategy of focused tOPV SIAs before OPV2 withdrawal in areas at risk of VDPV2 emergence and in sufficient number to raise population immunity

  16. Preventing Vaccine-Derived Poliovirus Emergence during the Polio Endgame.

    PubMed

    Pons-Salort, Margarita; Burns, Cara C; Lyons, Hil; Blake, Isobel M; Jafari, Hamid; Oberste, M Steven; Kew, Olen M; Grassly, Nicholas C

    2016-07-01

    Reversion and spread of vaccine-derived poliovirus (VDPV) to cause outbreaks of poliomyelitis is a rare outcome resulting from immunisation with the live-attenuated oral poliovirus vaccines (OPVs). Global withdrawal of all three OPV serotypes is therefore a key objective of the polio endgame strategic plan, starting with serotype 2 (OPV2) in April 2016. Supplementary immunisation activities (SIAs) with trivalent OPV (tOPV) in advance of this date could mitigate the risks of OPV2 withdrawal by increasing serotype-2 immunity, but may also create new serotype-2 VDPV (VDPV2). Here, we examine the risk factors for VDPV2 emergence and implications for the strategy of tOPV SIAs prior to OPV2 withdrawal. We first developed mathematical models of VDPV2 emergence and spread. We found that in settings with low routine immunisation coverage, the implementation of a single SIA increases the risk of VDPV2 emergence. If routine coverage is 20%, at least 3 SIAs are needed to bring that risk close to zero, and if SIA coverage is low or there are persistently "missed" groups, the risk remains high despite the implementation of multiple SIAs. We then analysed data from Nigeria on the 29 VDPV2 emergences that occurred during 2004-2014. Districts reporting the first case of poliomyelitis associated with a VDPV2 emergence were compared to districts with no VDPV2 emergence in the same 6-month period using conditional logistic regression. In agreement with the model results, the odds of VDPV2 emergence decreased with higher routine immunisation coverage (odds ratio 0.67 for a 10% absolute increase in coverage [95% confidence interval 0.55-0.82]). We also found that the probability of a VDPV2 emergence resulting in poliomyelitis in >1 child was significantly higher in districts with low serotype-2 population immunity. Our results support a strategy of focused tOPV SIAs before OPV2 withdrawal in areas at risk of VDPV2 emergence and in sufficient number to raise population immunity above the

  17. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

    PubMed Central

    Kamgoué, Alain; Murray, Heath; Pasta, Franck

    2016-01-01

    Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of

  18. Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1

    PubMed Central

    Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart

    2016-01-01

    ABSTRACT The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980T, suggesting their broad applicability as genetic tools for Natrinema species. IMPORTANCE Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980T. This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of

  19. Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

    PubMed

    Shen, Ling; Chen, Crystal Y; Huang, Dan; Wang, Richard; Zhang, Meihong; Qian, Lixia; Zhu, Yanfen; Zhang, Alvin Zhuoran; Yang, Enzhuo; Qaqish, Arwa; Chumakov, Konstantin; Kouiavskaia, Diana; Vignuzzi, Marco; Nathanson, Neal; Macadam, Andrew J; Andino, Raul; Kew, Olen; Xu, Junfa; Chen, Zheng W

    2017-07-15

    Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 10(7) to 10(9) 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 10(8) or 10(9) TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with

  20. Assessment of areas at increased risk for poliovirus circulation in Ecuador.

    PubMed

    Dayan, Gustavo H; Rodríguez, Rodrigo; Vinjé, Jan; Vásconez, Nancy; Cáceres, Víctor; Gregoricus, Nicole; Sobsey, Mark; Landaverde, Mauricio

    2004-10-01

    To assess areas at risk for poliovirus circulation in Ecuador, we first selected provinces at highest risk based on low immunization coverage with three doses of oral poliovirus vaccine, and a low number of reported cases of acute flaccid paralysis (AFP). Subsequently, we reviewed discharge data for the period 1996--2000 for diagnoses compatible with AFP in the only two national referral hospitals in Quito, and at least two main hospitals in each of the six selected provinces. Environmental samples from one or two cities/towns in each selected province were tested for poliovirus. Of the 14 identified AFP-compatible cases, 8 (57%) had been previously reported and investigated. We visited four out of the six unreported cases; none of those four had sequelae compatible with poliomyelitis. From the 14 environmental samples taken, we identified Sabin viruses in six of the samples; no vaccine-derived polioviruses were isolated. Using this methodology, we found no evidence of undetected poliovirus circulation in Ecuador.

  1. Innate host barriers to viral trafficking and population diversity: Lessons learned from poliovirus

    PubMed Central

    Pfeiffer, Julie K.

    2011-01-01

    Poliovirus is an error-prone enteric virus spread by the fecal-oral route, and rarely invades the central nervous system (CNS). However, in the rare instances when poliovirus invades the CNS, the resulting damage to motor neurons is striking and often permanent. In the pre-vaccine era, it is likely that most individuals within an epidemic community were infected; however, only 0.5% of infected individuals developed paralytic poliomyelitis. Paralytic poliomyelitis terrified the public and initiated a huge research effort, which was rewarded with two outstanding vaccines. During research to develop the vaccines, many questions were asked: Why did certain people develop paralysis? How does the virus move from the gut to the CNS? What limits viral trafficking to the CNS in the vast majority of infected individuals? Despite over 100 years of poliovirus research, many of these questions remain unanswered. The goal of this chapter is to review our knowledge of how poliovirus moves within and between hosts, how host barriers limit viral movement, how viral population dynamics impact viral fitness and virulence, and to offer hypotheses to explain the rare incidence of paralytic poliovirus disease. PMID:20951871

  2. An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

    PubMed Central

    Jenkins, O; Cason, J; Burke, K L; Lunney, D; Gillen, A; Patel, D; McCance, D J; Almond, J W

    1990-01-01

    It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed. Images PMID:2154604

  3. Environmental surveillance of wild poliovirus circulation in Egypt--balancing between detection sensitivity and workload.

    PubMed

    Hovi, Tapani; Blomqvist, Soile; Nasr, Eman; Burns, Cara C; Sarjakoski, Tarja; Ahmed, Nahed; Savolainen, Carita; Roivainen, Merja; Stenvik, Mirja; Laine, Pia; Barakat, Ibrahim; Wahdan, Mohammed H; Kamel, Faten A; Asghar, Humayun; Pallansch, Mark A; Kew, Olen M; Gary, Howard E; deGourville, Esther M; El Bassioni, Laila

    2005-06-01

    Examination of sewage specimens for poliovirus (environmental surveillance) was adopted as a supplementary tool in the surveillance of poliomyelitis in Egypt. Sewage samples were concentrated about 50-fold using a simple two-phase separation technique, and inoculated in cell cultures in two collaborating laboratories in parallel. All but 9 of the 293 (97%) samples collected from January 2001 to December 2002 contained poliovirus and/or other enteroviruses, with polioviruses being detected in 84% of the samples. The proportion of specimens containing type 1 wild poliovirus (PV1W, the North-East African (NEAF) genotype) was less in 2002 (16%) than in 2001 (57%), and further decreased in 2003. While the overall sensitivity to detect PV1W was similar in the two collaborating laboratories, the specimens scored positive were not identical. Parallel cultures inoculated with aliquots of a given specimen very frequently resulted in isolation of different viruses. Moreover, partial sequence analysis occasionally revealed representatives of different genetic lineages of PV1W in a given specimen. These results emphasize the need to use intensive laboratory analysis to optimise sample sensitivity in environmental poliovirus surveillance, and the difficulties in reproducing the isolation results by simple re-inoculation of samples containing a mixture of different viruses.

  4. Estimated Effect of Inactivated Poliovirus Vaccine Campaigns, Nigeria and Pakistan, January 2014–April 2016

    PubMed Central

    Shirreff, George; Wadood, Mufti Zubair; Vaz, Rui Gama; Sutter, Roland W.

    2017-01-01

    In 2014, inactivated poliovirus vaccine (IPV) campaigns were implemented in Nigeria and Pakistan after clinical trials showed that IPV boosts intestinal immunity in children previously given oral poliovirus vaccine (OPV). We estimated the effect of these campaigns by using surveillance data collected during January 2014–April 2016. In Nigeria, campaigns with IPV and trivalent OPV (tOPV) substantially reduced the incidence of poliomyelitis caused by circulating serotype-2 vaccine–derived poliovirus (incidence rate ratio [IRR] 0.17 for 90 days after vs. 90 days before campaigns, 95% CI 0.04–0.78) and the prevalence of virus in environmental samples (prevalence ratio [PR] 0.16, 95% CI 0.02–1.33). Campaigns with tOPV alone resulted in similar reductions (IRR 0.59, 95% CI 0.18–1.97; PR 0.45, 95% CI 0.21–0.95). In Pakistan, the effect of IPV+tOPV campaigns on wild-type poliovirus was not significant. Results suggest that administration of IPV alongside OPV can decrease poliovirus transmission if high vaccine coverage is achieved. PMID:27861118

  5. Translation without eIF2 promoted by poliovirus 2A protease.

    PubMed

    Redondo, Natalia; Sanz, Miguel Angel; Welnowska, Ewelina; Carrasco, Luis

    2011-01-01

    Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2A(pro) alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2A(pro) variant unable to cleave eIF4G. The level of 2A(pro) synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2A(pro) at high concentrations is necessary. The finding that 2A(pro) provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis.

  6. [Absence of poliovirus circulation in Colombian departments with vaccination coverage below 80%].

    PubMed

    González, María Mercedes; Sarmiento, Luis; Rey-Benito, Gloria Janneth; Padilla, Leonardo; Giraldo, Alejandra María; Castaño, Jhon Carlos

    2012-08-01

    This study aims to explore a possible silent circulation of wild and vaccine-derived polioviruses in departments of Colombia with polio vaccination coverage of below 80%. The study collected 52 samples of wastewater concentrated as a result of precipitation with polyethylene glycol and sodium chloride. The viral detection was carried out through isolation and the identification through neutralization of the cytopathic effect, as well as through a conventional polymerase chain reaction following reverse transcription. The isolated polioviruses were characterized by the VP1 gene sequence. In two of the 52 samples, there was a presence of the Sabin type 2 poliovirus with more than 99% sequence similarity with the Sabin type 2 strain polio. Circulation of the nonpolio enterovirus was detected in 17.3% of the samples. The serotypes identified corresponded to coxsackievirus B1, echovirus 30, and echovirus 11. No evidence of the spread of either vaccine-derived poliovirus or wild poliovirus was detected in the departments of Colombia with polio coverage lower than 80%.

  7. Public health response to the silent reintroduction of wild poliovirus to Israel, 2013-2014.

    PubMed

    Moran-Gilad, J; Kaliner, E; Gdalevich, M; Grotto, I

    2016-12-01

    During 2013/14, Israel witnessed the silent reintroduction and sustained transmission of wild poliovirus type 1 (WPV1) detected through routine environmental surveillance performed on sewage samples. The public health response to silent poliovirus transmission in a population with high inactivated polio vaccine (IPV) coverage poses an emerging challenge towards the 'End Game' of global poliovirus eradication. This paper reviews the risk assessment, risk management and risk communication aspects of this poliovirus incident. Special emphasis is placed on the use of scientific data generated in the risk assessment phase to inform the public health response. Reintroducing a live vaccine in supplemental immunization activities in response to transmission of WPV or vaccine-derived poliovirus should be considered close to the 'End Game' of polio eradication, especially if targeting the population at risk is feasible. Such circumstances require a comprehensive contingency plan that will support the generation of important public health evidence at the risk assessment stage, thereby allowing to tailor the risk management approaches and underpin appropriate risk communication.

  8. Assessment of areas at increased risk for poliovirus circulation in Ecuador.

    PubMed Central

    Dayan, Gustavo H.; Rodríguez, Rodrigo; Vinjé, Jan; Vásconez, Nancy; Cáceres, Víctor; Gregoricus, Nicole; Sobsey, Mark; Landaverde, Mauricio

    2004-01-01

    To assess areas at risk for poliovirus circulation in Ecuador, we first selected provinces at highest risk based on low immunization coverage with three doses of oral poliovirus vaccine, and a low number of reported cases of acute flaccid paralysis (AFP). Subsequently, we reviewed discharge data for the period 1996--2000 for diagnoses compatible with AFP in the only two national referral hospitals in Quito, and at least two main hospitals in each of the six selected provinces. Environmental samples from one or two cities/towns in each selected province were tested for poliovirus. Of the 14 identified AFP-compatible cases, 8 (57%) had been previously reported and investigated. We visited four out of the six unreported cases; none of those four had sequelae compatible with poliomyelitis. From the 14 environmental samples taken, we identified Sabin viruses in six of the samples; no vaccine-derived polioviruses were isolated. Using this methodology, we found no evidence of undetected poliovirus circulation in Ecuador. PMID:15473140

  9. Detection of vaccine-derived polioviruses in Mexico using environmental surveillance.

    PubMed

    Esteves-Jaramillo, Alejandra; Estívariz, Concepción F; Peñaranda, Silvia; Richardson, Vesta L; Reyna, Jesús; Coronel, Diana L; Carrión, Veronica; Landaverde, Jose M; Wassilak, Steven G F; Pérez-Sánchez, Elda E; López-Martínez, Irma; Burns, Cara C; Pallansch, Mark A

    2014-11-01

    Early detection and control of vaccine-derived poliovirus (VDPV) emergences are essential to secure the gains of polio eradication. Serial sewage samples were collected in 4 towns of Mexico before, throughout, and after the May 2010 oral poliovirus vaccine (OPV) mass immunization campaign. Isolation and molecular analysis of polioviruses from sewage specimens monitored the duration of vaccine-related strains in the environment and emergence of vaccine-derived polioviruses in a population partially immunized with inactivated poliovirus vaccine (IPV). Sabin strains were identified up to 5-8 weeks after the campaign in all towns; in Aguascalientes, 1 Sabin 3 was isolated 16 weeks after the campaign, following 7 weeks with no Sabin strains detected. In Tuxtla Gutiérrez, type 2 VDPV was isolated from 4 samples collected before and during the campaign, and type 1 VDPV from 1 sample collected 19 weeks afterward. During 2009-2010, coverage in 4 OPV campaigns conducted averaged only 57% and surveillance for acute flaccid paralysis (AFP) was suboptimal (AFP rate<1 per 100,000 population<15 years of age) in Tuxtla Gutierrez. VDPVs may emerge and spread in settings with inadequate coverage with IPV/OPV vaccination. Environmental surveillance can facilitate early detection in these settings. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. A Review of Historical Naked-Eye Sungrazing Comets

    NASA Astrophysics Data System (ADS)

    Knight, Matthew M.

    2013-10-01

    With the upcoming perihelion passage of Comet ISON (C/2012 S1) in November 2013, there is considerable interest in sungrazing comets at present. However, given the infrequency with which such comets appear, there have been few systematic studies of their behavior near perihelion. Bright, sometimes even daytime-observable comets have been recorded by observers around the world for more than 2000 years. A number of authors have compiled records of possible historical sungrazing comets, e.g., Hasegawa 1979, Kronk 1999, Hasegawa & Nakano 2001, England 2002, and Strom 2002. We review this literature to estimate the frequency of arrival of naked-eye sungrazing comets and investigate if there are any trends in their behavior, such as between peak brightness and length of time a comet is observable. We will also review the “modern” observations of naked-eye sungrazing comets (primarily Kreutz group comets observed since 1800) to investigate the frequency of fragmentation near the Sun and the dependence of survivability on size (as inferred from brightness) and/or perihelion distance.

  11. Naked-eye sensor for rapid determination of mercury ion.

    PubMed

    Liu, Jing; Wu, Dapeng; Yan, Xiaohui; Guan, Yafeng

    2013-11-15

    A naked-eye paper sensor for rapid determination of trace mercury ion in water samples was designed and demonstrated. The mercury-sensing rhodamine B thiolactone was immobilized in silica matrices and the silica matrices were impregnated firmly and uniformly in the filter paper. As water samples flow through the filter paper, the membrane color will change from white to purple red, which could be observed obviously with naked eye, when concentration of mercury ions equals to or exceeds 10nM, the maximum residue level in drinking water recommended by U.S. EPA. The color change can also be recorded by a flatbed scanner and then digitized, reducing the detection limit of Hg(2+) down to 1.2 nM. Moreover, this method is extremely specific for Hg(2+) and shows a high tolerance ratio of interferent coexisting ions. The presence of Na(+) (2 mM), K(+) (2 mM), Fe(3+) (0.1 mM), Zn(2+) (0.1 mM), Mg(2+) (0.1 mM), Ni(2+) (50 μM), Co(2+) (50 μM), Cd(2+) (50 μM), Pb(2+) (50 μM), Cu(2+) (50 μM) and Ag(+) (3.5 μM) did not interfere with the detection of Hg(2+) (25 nM). Finally, the present method was applied in the detection of Hg(2+) in mineral water, tap water and pond water.

  12. Sensitive, Quantitative Naked-Eye Biodetection with Polyhedral Cu Nanoshells.

    PubMed

    Kim, Jae-Ho; Park, Jeong-Eun; Lin, Mouhong; Kim, Sungi; Kim, Gyeong-Hwan; Park, SungJun; Ko, GwangPyo; Nam, Jwa-Min

    2017-10-01

    One of the most heavily used methods in chemical and biological labeling, detection, and imaging is based on silver shell-based enhancement on Au nanoparticles (AuNPs) that is useful for amplifying Rayleigh scattering, colorimetric signal, surface-enhanced Raman scattering, and electrical signal, but poor structural controllability and nonspecific growth of silver shells have limited its applications, especially with respect to signal reproducibility and quantification. Here, a highly specific, well-defined Cu nanopolyhedral shell overgrowth chemistry is developed with the aid of polyethyleneimine (PEI) on AuNPs, and the use of this PEI-mediated Cu polyhedral nanoshell (CuP) chemistry is shown as a means of light-scattering signal enhancement for the development of naked-eye-based highly sensitive and quantitative detections of DNA and viruses. Remarkably, these CuPs are exclusively formed on AuNPs in a controllable manner, with no noticeable nonspecific CuP growth. The findings enable to acquire clearly visible signals without analytic instrumentation, detectable down to 8 × 10(-15) m of DNA (anthrax sequence) and 2700 copies of viruses (noroviruses in clinical stool samples) with broad dynamic ranges on archetypal assay platforms. This new method provides a general platform in controlling Cu shell nanostructures and their optical signals, and opens up revenues for highly reliable, quantitative onsite naked-eye biodetection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    PubMed

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

  14. Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster ( Crassostrea gigas) and culture broth under different conditions

    NASA Astrophysics Data System (ADS)

    Jung, Pil-Mun; Park, Jae Seok; Park, Jin-Gyu; Park, Jae-Nam; Han, In-Jun; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Baek, Min; Chung, Young-Jin; Lee, Ju-Woon

    2009-07-01

    Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster ( Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation.

  15. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat.

    PubMed

    Tian, Xiao; Azpurua, Jorge; Hine, Christopher; Vaidya, Amita; Myakishev-Rempel, Max; Ablaeva, Julia; Mao, Zhiyong; Nevo, Eviatar; Gorbunova, Vera; Seluanov, Andrei

    2013-07-18

    The naked mole rat (Heterocephalus glaber) displays exceptional longevity, with a maximum lifespan exceeding 30 years. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years. In addition to their longevity, naked mole rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer. Here we identify a mechanism responsible for the naked mole rat's cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This high-molecular-mass HA accumulates abundantly in naked mole-rat tissues owing to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signalling, as they have a higher affinity to HA compared with mouse or human cells. Perturbation of the signalling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high-molecular-mass HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, HYAL2, naked mole-rat cells become susceptible to malignant transformation and readily form tumours in mice. We speculate that naked mole rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

  16. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis.

    PubMed

    Lindsey, Rebecca L; Frye, Jonathan G; Thitaram, Sutawee N; Meinersmann, Richard J; Fedorka-Cray, Paula J; Englen, Mark D

    2011-06-01

    The objective of this study was to examine the distribution of multidrug resistance in Escherichia coli in relation to plasmid replicon types, animal sources, and genotypes. E. coli isolates (n = 35) from seven different animal sources were selected and tested for susceptibility to 15 antimicrobials; pulsed-field gel electrophoresis was used to determine genetic relationships among the E. coli isolates. Plasmid types based on their incompatibility (Inc) replicon types were determined, and linkage disequilibrium analysis was performed for antimicrobial resistance profiles, replicon types, and animal source. A high degree of genotypic diversity was observed: 34 different pulsed-field gel electrophoresis types among the 35 isolates examined. Twelve different plasmid Inc types were detected, and all isolates carried at least one replicon type. IncF (n = 25; 71.4%) and IncFIB (n = 19; 54.3%) were the most common replicon types identified. Chloramphenicol resistance was significantly linked with four Inc types (A/C, FIIA, F, and Y), and amoxicillin/clavulanic acid was linked with three Inc types (B/O, P and Y). Resistance to any other antimicrobial was linked to two or fewer replicon types. The isolate source was linked with resistance to seven antimicrobials and IncI1. We conclude that commensal E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance.

  17. Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).

    PubMed

    Olsen, Christel M; Pemula, Anand Kumar; Braaen, Stine; Sankaran, Krishnan; Rimstad, Espen

    2013-11-19

    The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.

  18. The Practical Consideration of Poliovirus as an Oncolytic Virotherapy

    PubMed Central

    Denniston, Elizabeth; Crewdson, Hannah; Rucinsky, Nicole; Stegman, Andrew; Remenar, Diana; Moio, Katherine; Clark, Brianne; Higginbotham, Alexandra; Keffer, Ross; Brammer, Sarah; Horzempa, Joseph

    2017-01-01

    The inauguration of novel treatment strategies into the clinical setting faces a number of hurdles. In addition to treatment efficacy and safety, acceptance by doctors and patients is paramount to the success of novel therapies. Although viruses are the cause of numerous infectious diseases, these acellular entities have been harnessed over the years to benefit mankind. Recently, a recombinant Poliovirus-Rhinovirus Chimera (PVSRIPO) has shown promise for the treatment of glioblastoma in clinical trials as well as other cancer types in animal models. In this literature review, we discuss the use of PVSRIPO as an oncolytic virotherapy. In addition to being a potential treatment for glioblastoma, this recombinant virus could possibly be used against other cancers because many tumor cells express the PVSRIPO receptor antigens (CD155) and have a limited ability to control viral replication. Moreover, virus-induced immune responses contribute to the efficacy of PVSRIPO. Given the current trajectory of this experimental therapy, the possibility exists that PVSRIPO will soon be a viable treatment option for various cancer types. While many healthcare providers and cancer patients likely welcome this new viral based treatment, history has taught us that some may be skeptical and avoid its use because of the viral composition of this therapy. PMID:28203321

  19. Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral poliovirus vaccine quality control.

    PubMed

    Rezapkin, G V; Norwood, L P; Taffs, R E; Dragunsky, E M; Levenbook, I S; Chumakov, K M

    1995-08-20

    Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U-->C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be

  20. A topoisomerase II-dependent mechanism for resetting replicons at the S–M-phase transition

    PubMed Central

    Cuvier, Olivier; Stanojcic, Slavica; Lemaitre, Jean-Marc; Mechali, Marcel

    2008-01-01

    Topoisomerase II (topo II) is required for chromosome segregation and for reprogramming replicons. Here, we show that topo II couples DNA replication termination with the clearing of replication complexes for resetting replicons at mitosis. Topo II inhibition impairs completion of DNA replication, accounting for replication protein A (RPA) stabilization onto ssDNA. Topo II inhibition does not affect the caffeine-sensitive ORC1 degradation found upon origin firing, but it impairs the cdk-dependent degradation/chromatin dissociation of an ORC1/2 reservoir at mitosis. Our results show that ORC1 degradation is rescued by Pin1 depletion and that this topo II-dependent clearing of ORC1/2 from chromatin involves the APC. PMID:18381889

  1. Mapping of active replication origins in vivo in thaum- and euryarchaeal replicons.

    PubMed

    Pelve, Erik A; Martens-Habbena, Willm; Stahl, David A; Bernander, Rolf

    2013-11-01

    We report mapping of active replication origins in thaum- and euryarchaeal replicons using high-throughput sequencing-based marker frequency analysis. The chromosome of the thaumarchaeon Nitrosopumilus maritimus is shown to contain a single origin of replication, whereas the main chromosome in the halophilic euryarchaea Haloferax mediterranei and Haloferax volcanii each contains two origins. All replication origins specified bidirectional replication, and the two origins in the halophiles were initiated in synchrony. The pHM500 plasmid of H. mediterranei is shown to contain a single origin, and the copy numbers of five plasmid replicons in the two halophiles were inferred to be close to that of the main chromosome. Origin recognition boxes (ORBs) that provide binding sites for Orc1/Cdc6 replication initiator proteins are identified at all chromosomal origins, as well as in a range of additional thaumarchaeal species. An annotation update is provided for all three species. © 2013 John Wiley & Sons Ltd.

  2. Poliovirus surveillance by examining sewage specimens. Quantitative recovery of virus after introduction into sewerage at remote upstream location.

    PubMed

    Hovi, T; Stenvik, M; Partanen, H; Kangas, A

    2001-08-01

    In order to assess the feasibility of environmental poliovirus surveillance, known amounts of poliovirus type 1, strain Sabin, were flushed into the sewage network of Helsinki. Grab specimens collected at a remote downstream location and concentrated about a 100-fold revealed infectious poliovirus on four successive days in all three separate experiments. As for concentration, a simple two-phase separation method was found to be at least as useful as a several-fold more resource-demanding polyethylene glycol (PEG) precipitation method. Recovery of the introduced virus was remarkably high (more than 10%). Using the current system, it might be possible to detect poliovirus circulation in a population of 700,000 people by examining a single 400 ml sewage specimen, if 1 out of 10,000 inhabitants were excreting the virus. It is concluded that environmental surveillance is a sensitive approach to monitor silent poliovirus circulation in populations served by a sewage network.

  3. Progress in the development of poliovirus antiviral agents and their essential role in reducing risks that threaten eradication.

    PubMed

    McKinlay, Mark A; Collett, Marc S; Hincks, Jeffrey R; Oberste, M Steven; Pallansch, Mark A; Okayasu, Hiromasa; Sutter, Roland W; Modlin, John F; Dowdle, Walter R

    2014-11-01

    Chronic prolonged excretion of vaccine-derived polioviruses by immunodeficient persons (iVDPV) presents a personal risk of poliomyelitis to the patient as well as a programmatic risk of delayed global eradication. Poliovirus antiviral drugs offer the only mitigation of these risks. Antiviral agents may also have a potential role in the management of accidental exposures and in certain outbreak scenarios. Efforts to discover and develop poliovirus antiviral agents have been ongoing in earnest since the formation in 2007 of the Poliovirus Antivirals Initiative. The most advanced antiviral, pocapavir (V-073), is a capsid inhibitor that has recently demonstrated activity in an oral poliovirus vaccine human challenge model. Additional antiviral candidates with differing mechanisms of action continue to be profiled and evaluated preclinically with the goal of having 2 antivirals available for use in combination to treat iVDPV excreters.

  4. Poliovirus surveillance by examining sewage specimens. Quantitative recovery of virus after introduction into sewerage at remote upstream location.

    PubMed Central

    Hovi, T.; Stenvik, M.; Partanen, H.; Kangas, A.

    2001-01-01

    In order to assess the feasibility of environmental poliovirus surveillance, known amounts of poliovirus type 1, strain Sabin, were flushed into the sewage network of Helsinki. Grab specimens collected at a remote downstream location and concentrated about a 100-fold revealed infectious poliovirus on four successive days in all three separate experiments. As for concentration, a simple two-phase separation method was found to be at least as useful as a several-fold more resource-demanding polyethylene glycol (PEG) precipitation method. Recovery of the introduced virus was remarkably high (more than 10%). Using the current system, it might be possible to detect poliovirus circulation in a population of 700,000 people by examining a single 400 ml sewage specimen, if 1 out of 10,000 inhabitants were excreting the virus. It is concluded that environmental surveillance is a sensitive approach to monitor silent poliovirus circulation in populations served by a sewage network. PMID:11561962

  5. Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

    PubMed

    Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Tian, Chang Fu; Chen, Wen Xin

    2014-02-01

    In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

  6. Three replicons of Rhizobium sp. Strain NGR234 harbor symbiotic gene sequences.

    PubMed

    Flores, M; Mavingui, P; Girard, L; Perret, X; Broughton, W J; Martínez-Romero, E; Dávila, G; Palacios, R

    1998-11-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.

  7. Replicon-Dependent Differentiation of Symbiosis-Related Genes in Sinorhizobium Strains Nodulating Glycine max

    PubMed Central

    Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Chen, Wen Xin

    2014-01-01

    In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons. PMID:24317084

  8. Three Replicons of Rhizobium sp. Strain NGR234 Harbor Symbiotic Gene Sequences

    PubMed Central

    Flores, Margarita; Mavingui, Patrick; Girard, Lourdes; Perret, Xavier; Broughton, William J.; Martínez-Romero, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1998-01-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b. PMID:9811668

  9. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

    PubMed

    Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J; Plante, Jessica A; Frolova, Elena I; Beasley, David W C; Frolov, Ilya

    2014-07-22

    Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

  10. Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication

    PubMed Central

    Dotti, Silvia; Lombardo, Tina; Villa, Riccardo; Cacciamali, Andrea; Zanotti, Cinzia; Andreani, Nadia Andrea; Cinotti, Stefano; Ferrari, Maura

    2017-01-01

    The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field. PMID:28046048

  11. [The role of Sabin inactivated poliovirus vaccine in the final phase of global polio eradication].

    PubMed

    Dong, S Z; Zhu, W B

    2016-12-06

    Global polio eradication has entered its final phase, but still faces enormous challenges. The Polio Eradication and Endgame Strategic Plan (2013-2018) set the target for making the world polio-free by 2018. Meanwhile, the World Heath Organization Global Action Plan (GAP Ⅲ) recommended that polioviruses be stored under strict conditions after eradication of the wild poliovirus. At least one dose of inactivated poliovirus vaccine (IPV) would be required for each newborn baby in the world to ensure successful completion of the final strategy and GAP Ⅲ. The Sabin IPV has a high production safety and low production cost, compared with the wild-virus IPV and, therefore, can play an important role in the final stage of global polio eradication.

  12. New Generation of Inactivated Poliovirus Vaccines for Universal Immunization After Eradication of Poliomyelitis

    PubMed Central

    Chumakov, Konstantin; Ehrenfeld, Ellie

    2008-01-01

    Twenty years of global polio eradication efforts may soon eliminate wild-type poliovirus transmission. However, new information about poliovirus learned during this campaign, as well as the political realities of a modern world demand that universal immunity against poliomyelitis be maintained even after wild poliovirus is eradicated. Although two excellent vaccines have proven highly effective in the past, neither the live nor current inactivated products are optimal for use in the post-eradication setting. Therefore, concerted efforts are urgently needed to develop a new generation of vaccine that is risk-free and affordable and can be produced on a global scale. Here we discuss the desired properties and ways to create a new polio vaccine. PMID:18990066

  13. Outbreak of type 1 wild poliovirus infection in adults, Namibia, 2006.

    PubMed

    Yusuf, Nasir; de Wee, Rosalina; Foster, Norbert; Watkins, Margaret A; Tiruneh, Desta; Chauvin, Claire; Bossarte, Robert; Mandlhate, Custodia; Jack, Abdoulie; Gumede, Nicksy; Mawela, Alfred; Burns, Cara C; Pallansch, Mark A; Allies, Tina; Rainey, Jeannette; Mataruse, Noah; Nshimirimana, Deo

    2014-11-01

    A paralytic poliomyelitis outbreak occurred in Namibia in 2006, almost exclusively among adults. Nineteen cases were virologically confirmed as due to wild poliovirus type 1 (WPV1), and 26 were classified as polio compatible. Eleven deaths occurred among confirmed and compatible cases (24%). Of the confirmed cases, 97% were aged 15-45 years, 89% were male, and 71% lived in settlement areas in Windhoek. The virus was genetically related to a virus detected in 2005 in Angola, which had been imported earlier from India. The outbreak is likely due to immunity gaps among adults who were inadequately vaccinated during childhood. This outbreak underscores the ongoing risks posed by poliovirus importations, the importance of maintaining strong acute flaccid paralysis surveillance even in adults, and the need to maintain high population immunity to avoid polio outbreaks in the preeradication period and outbreaks due to vaccine-derived polioviruses in the posteradication era.

  14. Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

    PubMed

    Bujaki, Erika

    2016-01-01

    The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

  15. Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

    PubMed Central

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M.; Frolov, Ilya

    2005-01-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5′ untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins. PMID:15919912

  16. Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells

    PubMed Central

    Frolov, Ilya; Agapov, Eugene; Hoffman, Thomas A.; Prágai, Béla M.; Lippa, Mara; Schlesinger, Sondra; Rice, Charles M.

    1999-01-01

    The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989–12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments. PMID:10196280

  17. Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    PubMed Central

    Forrest, Sophie; Lear, Zoe; Herod, Morgan R.; Ryan, Martin; Rowlands, David J.

    2014-01-01

    We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2′-C-methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future. PMID:25096816

  18. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells.

    PubMed

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M; Frolov, Ilya

    2005-06-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

  19. Naked singularities in non-self-similar gravitational collapse of radiation shells

    SciTech Connect

    Joshi, P.S.; Dwivedi, I.H. )

    1992-03-15

    Non-self-similar gravitational collapse of imploding radiation is shown to give rise to a strong curvature naked singularity. The conditions are specified for the singularity to be globally naked and the strength of the same is examined along nonspacelike curves and along all the families of nonspacelike geodesics terminating at the singularity in the past.

  20. The Naked Mole Rat Genome Resource: facilitating analyses of cancer and longevity-related adaptations

    PubMed Central

    Keane, Michael; Craig, Thomas; Alföldi, Jessica; Berlin, Aaron M.; Johnson, Jeremy; Seluanov, Andrei; Gorbunova, Vera; Di Palma, Federica; Lindblad-Toh, Kerstin; Church, George M.; de Magalhães, João Pedro

    2014-01-01

    Motivation: The naked mole rat (Heterocephalus glaber) is an exceptionally long-lived and cancer-resistant rodent native to East Africa. Although its genome was previously sequenced, here we report a new assembly sequenced by us with substantially higher N50 values for scaffolds and contigs. Results: We analyzed the annotation of this new improved assembly and identified candidate genomic adaptations which may have contributed to the evolution of the naked mole rat’s extraordinary traits, including in regions of p53, and the hyaluronan receptors CD44 and HMMR (RHAMM). Furthermore, we developed a freely available web portal, the Naked Mole Rat Genome Resource (http://www.naked-mole-rat.org), featuring the data and results of our analysis, to assist researchers interested in the genome and genes of the naked mole rat, and also to facilitate further studies on this fascinating species. Availability and implementation: The Naked Mole Rat Genome Resource is freely available online at http://www.naked-mole-rat.org. This resource is open source and the source code is available at https://github.com/maglab/naked-mole-rat-portal. Contact: jp@senescence.info PMID:25172923

  1. Relative sensitivity of three cell substrates to the Sabin poliovirus strains.

    PubMed

    Kado, G

    Poliovirus types 1, 2 and 3 (Sabin strains) were titrated using microtissue culture plates in three different cell substrates: Hep 2C, Vero and MRC-5 human diploid cells. Each type of poliovirus was titrated 60 times. The virus dilutions were inoculated in the three cell substrates and the titres (TCID50/ml) calculated by the Spearman-Kärber method. The results were analyzed by the U-test (Mann and Whitney procedure) in a Wang 600 computer. For poliovirus type 1 the differences in titres observed between Hep 2C and Vero cells were not statistically significant at p = 0.01, but the differences in titres between Hep 2C and MRC-5 human diploid cells and between Vero and MRC-5 cells were statistically highly significant (p = 0.001). For poliovirus types 2 and 3 the observed differences in titres were highly significant (p = 0.001) between Hep 2C and Vero cells, Hep 2C and MRC-5 and also between Vero and MRC-5 cells, with higher titres on Hep 2C cells. Accordingly with these results Hep 2C and Vero cells showed the same susceptibility to poliovirus type 1. In MCR-5 cells the average titre (G.M.T.) was 1 log10 below the average titres (G.M.T.) from Hep 2C or Vero cells. With poliovirus types 2 and 3 the higher titres were obtained on Hep 2C cells with significant differences to the titres observed in Vero and MRC-5 human diploid cells, which showed less susceptibility.

  2. Isolation of sabin-like polioviruses from wastewater in a country using inactivated polio vaccine.

    PubMed

    Zurbriggen, Sebastian; Tobler, Kurt; Abril, Carlos; Diedrich, Sabine; Ackermann, Mathias; Pallansch, Mark A; Metzler, Alfred

    2008-09-01

    From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5' untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

  3. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

    PubMed

    Sanders, Barbara P; de Los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G; Song, Yutong; Cooper, Gillian; Crawt, Laura E; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-03-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  4. Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

    PubMed Central

    Sanders, Barbara P.; de los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G.; Song, Yutong; Cooper, Gillian; Crawt, Laura E.; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H. H. V.; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

    2016-01-01

    The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive

  5. Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro.

    PubMed

    Novoa, I; Feduchi, E; Carrasco, L

    1994-11-21

    Two hybrid proteins between Pseudomonas aeruginosa exotoxin A (PE) and poliovirus protease 2Apro have been generated. One hybrid protein contains the poliovirus 2Apro sequence replacing the region of PE corresponding to amino acids 413-607. The other hybrid contains in addition the transforming growth factor sequence. The two hybrid proteins were efficiently synthesized in E. coli cells using the inducible pET vectors. Both hybrid toxins cleaved p220 (eIF-4 gamma) when the recombinant plasmids were transfected in COS cells infected with recombinant vaccinia virus bearing the T7 RNA polymerase gene.

  6. Molecular evidence that Reticulomyxa filosa is a freshwater naked foraminifer.

    PubMed

    Pawlowski, J; Bolivar, I; Fahrni, J F; De Vargas, C; Bowser, S S

    1999-01-01

    Reticulomyxa filosa is a freshwater protist possessing fine granular, branching and anastomosing pseudopodia and therefore traditionally placed in the class Granuloreticulosea, order Athalamida, as a sister group to the order Foraminiferida. Recent studies have revealed remarkable similarities in pseudopodial motility and ultrastructure between R. filosa and foraminifera (e.g. Allogromia laticollaris), prompting us to conduct a molecular phylogenetic analysis of these seemingly disparate organisms. We sequenced the complete small-subunit of the ribosomal DNA of the cultured strain of R. filosa and compared it to the corresponding sequences of other protists including 12 species of foraminifera. We also sequenced and analyzed the actin coding genes from R. filosa and two species of foraminifera, Allogromia sp. and Ammonia sp. The analysis of both data sets clearly shows that R. filosa branches within the clade of foraminifera, suggesting that R. filosa is in fact a freshwater naked foraminiferan.

  7. The Poliovirus Empty Capsid Specifically Recognizes the Poliovirus Receptor and Undergoes Some, but Not All, of the Transitions Associated with Cell Entry

    PubMed Central

    Basavappa, Ravi; Gómez-Yafal, Alicia; Hogle, James M.

    1998-01-01

    Experimental results presented here demonstrate that the poliovirus empty capsid binds with saturable character to poliovirus-susceptible cells, binds preferentially to susceptible cells, and competes with mature virus for binding sites on cells. Hence, induced changes in the structure and/or stability of the particle by RNA encapsidation and virus maturation are not necessary for recognition by receptor. In mature virus, heat-induced rearrangements mimic those induced by receptor at physiological temperatures in several important respects, namely, expulsion of VP4 and externalization of the VP1 N-terminal arm. It is shown here that in the empty capsid the VP1 N-terminal arm is externalized but the VP4 portion of VP0 is not. Thus, these two hallmark rearrangements associated with cell entry can be uncoupled. PMID:9696852

  8. Development of real-time PCR to detect oral vaccine-like poliovirus and its application to environmental surveillance.

    PubMed

    Iwai-Itamochi, Masae; Yoshida, Hiromu; Obara-Nagoya, Mayumi; Horimoto, Eiji; Kurata, Takeshi; Takizawa, Takenori

    2014-01-01

    In order to perform environmental surveillance to track oral poliovirus vaccine-like poliovirus sensitively and conveniently, real-time PCR was developed and applied to a raw sewage concentrate. The real-time PCR method detected 0.01-0.1 TCID50 of 3 serotypes of Sabin strain specifically. The method also detected the corresponding serotypes of oral poliovirus vaccine-like poliovirus specifically, but detected neither wild poliovirus, except Mahoney for type 1 and Saukett for type 3, nor other enteric viruses, as far as examined. When real-time PCR was applied to environmental surveillance, the overall agreement rates between real-time PCR and the cell culture were 83.3% for all serotypes. Since real-time PCR has the advantages of rapid detection of viruses and minimum requirement of sampling volume as compared with ordinary cell culture, it is suitable to monitor oral poliovirus vaccine-like poliovirus in the environment, especially in areas where an oral vaccine is being replaced by an inactivated vaccine.

  9. Environmental surveillance of poliovirus and non-polio enterovirus in urban sewage in Dakar, Senegal (2007-2013).

    PubMed

    Ndiaye, Abdou Kader; Diop, Pape Amadou Mbathio; Diop, Ousmane Madiagne

    2014-01-01

    Global poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus in a population, and (ii) vaccination to prevent its diffusion. However, as poliovirus can survive in the environment namely in sewage, environmental surveillance (ES) is of growing importance as the eradication target is close. This study aimed to assess polioviruses and non polio enteroviruses circulation in sewage drains covering a significant population of Dakar. From April 2007 to May 2013, 271 specimens of raw sewage were collected using the grab method in 6 neighborhoods of Dakar. Samples were processed to extract and concentrate viruses using polyethylene glycol and Dextran (two-phase separation method). Isolation of enteroviruses was attempted in RD, L20B and Hep2 cell lines. Polioviruses were identified by RT-PCR and Elisa. Non Polio Enteroviruses (NPEVs) were identified by RT-PCR and microneutralisation tests. Polioviruses and NPEVs were respectively detected in 34,3% and 42,8% sewage samples. No wild poliovirus neither circulating vaccine-derived Poliovirus (cVDPV) was detected. Neutralization assays have identified 49 non polio enteroviruses that were subsequently classified in 13 serotypes belonging to HEV-A (22, 4%), HEV-B (12, 24%), HEV-C (26, 53%) and HEV-D (6, 12%) species. This study is the first documentation of enteroviruses environmental detection in Senegal. It shows the usefulness of environmental surveillance for indirect monitoring of the circulation and distribution of enteroviruses in the community.

  10. A Novel Adenoviral Hybrid-vector System Carrying a Plasmid Replicon for Safe and Efficient Cell and Gene Therapeutic Applications.

    PubMed

    Voigtlander, Richard; Haase, Rudolf; Mück-Hausl, Martin; Zhang, Wenli; Boehme, Philip; Lipps, Hans-Joachim; Schulz, Eric; Baiker, Armin; Ehrhardt, Anja

    2013-04-02

    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.

  11. Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber).

    PubMed

    Park, Thomas J; Lu, Ying; Jüttner, René; Smith, Ewan St J; Hu, Jing; Brand, Antje; Wetzel, Christiane; Milenkovic, Nevena; Erdmann, Bettina; Heppenstall, Paul A; Laurito, Charles E; Wilson, Steven P; Lewin, Gary R

    2008-01-01

    In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes "normal" mammalian nociception.

  12. Selective Inflammatory Pain Insensitivity in the African Naked Mole-Rat (Heterocephalus glaber)

    PubMed Central

    Park, Thomas J; Hu, Jing; Brand, Antje; Wetzel, Christiane; Milenkovic, Nevena; Erdmann, Bettina; Heppenstall, Paul A; Laurito, Charles E; Wilson, Steven P; Lewin, Gary R

    2008-01-01

    In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception. PMID:18232734

  13. Characterization of Poliovirus Variants Selected for Resistance to the Antiviral Compound V-073

    PubMed Central

    Liu, Hong-Mei; Roberts, Jason A.; Moore, Deborah; Anderson, Barbara; Pallansch, Mark A.; Pevear, Daniel C.; Collett, Marc S.

    2012-01-01

    V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n = 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n = 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073. PMID:22890765

  14. Update on Vaccine-Derived Polioviruses - Worldwide, January 2015-May 2016.

    PubMed

    Jorba, Jaume; Diop, Ousmane M; Iber, Jane; Sutter, Roland W; Wassilak, Steven G; Burns, Cara C

    2016-08-05

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4).

  15. Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis

    SciTech Connect

    Power, U.F.; Collins, J.K. )

    1989-06-01

    The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.

  16. Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli

    SciTech Connect

    Plotch, S.J.; Palant, O.; Gluzman, Y.

    1989-01-01

    A cDNA clone encoding the RNA polymerase of poliovirus has been expressed in Escherichia coli under the transcriptional control of a T7 bacteriophage promoter. This poliovirus enzyme was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant enzyme has been purified to near homogeneity, and polyclonal antibodies have been prepared against it. The enzyme exhibits poly(A)-dependent oligo(U)-primed ply(U) polymerase activity as well as RNA polymerase activity. In the presence of an oligo(U) primer, the enzyme catalyzes the synthesis of a full-length copy of either poliovirus or globin RNA templates. In the absence of added primer, RNA products up to twice the length of the template are synthesized. When incubated in the presence of a single nucleoside triphosphate, (..cap alpha..-/sup 32/P)UTP, the enzyme catalyzes the incorporation of radioactive label into template RNA. These results are discussed in light of previously proposed models of poliovirus RNA synthesis in vitro.

  17. [Eradication of poliomyelitis and emergence of pathogenic vaccine-derived polioviruses: from Madagascar to Cameroon].

    PubMed

    Delpeyroux, Francis; Colbère-Garapin, Florence; Razafindratsimandresy, Richter; Sadeuh-Mba, Serge; Joffret, Marie-Line; Rousset, Dominique; Blondel, Bruno

    2013-11-01

    The oral poliovaccine, a live vaccine made of attenuated poliovirus strains, is the main tool of the vaccination campaigns organised for eradicating poliomyelitis. these campaigns had led to the decline and, thereafter, to the disappearance of wild poliovirus strains of the three serotypes (1-3) in most parts of the world. However, when the poliovaccine coverage becomes too low, vaccine polioviruses can circulate in insufficiently immunized populations and become then pathogenic by mutations and genetic recombination with other enteroviruses of the same species, in particular some coxsackievirus A. These mutated and recombinant vaccine strains have been implicated in several epidemics of paralytic poliomyelitis. Two polio outbreaks associated with these pathogenic circulating vaccine-derived poliovirus (cVDPV) occurred in 2001-2002 and 2005 in the South of Madagascar where vaccine coverage was low. These cVDPV, of serotype 2 or 3, were isolated from paralyzed children and some of their healthy contacts. Other cVDPV were isolated in the same region from healthy children in 2011, indicating that these viruses were circulating again. Vaccination campaigns could stop the outbreaks in 2002 and 2005, and most probably prevent another one in 2011. Therefore, the genetic plasticity of poliovaccine strains that threatens the benefit of vaccination campaigns is the target of an accurate surveillance and an important theme of studies in the virology laboratories of the Institut Pasteur international network.

  18. Oncolytic virotherapy for human bone and soft tissue sarcomas using live attenuated poliovirus.

    PubMed

    Atsumi, Satoru; Matsumine, Akihiko; Toyoda, Hidemi; Niimi, Rui; Iino, Takahiro; Nakamura, Tomoki; Matsubara, Takao; Asanuma, Kunihiro; Komada, Yoshihiro; Uchida, Atsumasa; Sudo, Akihiro

    2012-09-01

    The poliovirus receptor CD155, is essential for poliovirus to infect and induce death in neural cells. Recently, CD155 has been shown to be selectively expressed on certain types of tumor cells originating from the neural crest, including malignant glioma and neuroblastoma. However, the expression pattern of CD155 in soft tissue sarcoma has not been examined. Therefore, we first examined CD155 expression in sarcoma cell lines, and found the expression of both CD155 mRNA and protein in 12 soft and bone tissue sarcoma cell lines. Furthermore, we examined the effect of live attenuated poliovirus (LAPV) on 6 bone and soft tissue sarcoma cell lines in vitro, and found that LAPV induced apoptosis by activating caspases 7 and 3 in all of these cell lines. Furthermore, in BALB/c nu/nu mice xenotransplanted with HT1080 fibrosarcoma cells, administration of live attenuated poliovirus caused growth suppression of the tumors. These results suggest that oncolytic therapy using a LAPV may represent a new option for the treatment of bone and soft tissue sarcomas.

  19. Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug.

    PubMed Central

    Fox, M P; Otto, M J; McKinlay, M A

    1986-01-01

    WIN 51711, a potent new antipicornavirus drug, has been shown to inhibit an early event in the replication cycle of human poliovirus type 2 and human rhinovirus type 2. WIN 51711 was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labeled virions to cells. When virion penetration of the plasma membrane was determined through loss of sensitivity to neutralizing antisera, WIN 51711 had no effect on poliovirus penetration, but inhibited rhinovirus penetration by 40%. In the presence of WIN 51711, exposure of neutral red-encapsidated virus-infected cells to light at 3 h postinfection resulted in a 3-log reduction in the number of infectious centers, indicating that WIN 51711 maintained the viral RNA in the encapsidated state after penetrating the cell membrane. The inhibition of uncoating by WIN 51711 in the neutral red assay was found to be concentration dependent, with a concentration of 0.03 micrograms/ml resulting in a 90% inhibition of uncoating. Sucrose gradient sedimentation of lysates from whole cells infected with [3H]uridine-labeled poliovirus showed that poliovirions remained intact in the presence of WIN 51711, but were uncoated in the absence of drug. WIN 51711 also prevented thermal inactivation of poliovirus infectivity, indicating a direct stabilizing effect of this compound on virion capsid conformation. PMID:3019232

  20. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  1. Vaccine-Derived Polioviruses and Children with Primary Immunodeficiency, Iran, 1995–2014

    PubMed Central

    Shaghaghi, Mohammadreza; Shahmahmoodi, Shohreh; Abolhassani, Hassan; Soleyman-jahi, Saeed; Parvaneh, Leila; Mahmoudi, Sussan; Chavoshzadeh, Zahra; Yazdani, Reza; Zahraei, Seyed Mohsen; Ebrahimi, Mohsen; Eslamian, Mohammad H.; Tabatabaie, Hamideh; Yousefi, Maryam; Kandelousi, Yaghoob M.; Oujaghlou, Aliasghar; Rezaei, Nima

    2016-01-01

    Widespread use of oral poliovirus vaccine has led to an ≈99.9% decrease in global incidence of poliomyelitis (from ≈350,000 cases in 1988 to 74 cases in 2015) and eradication of wild-type poliovirus serotypes 2 and 3. However, patients with primary immunodeficiency might shed vaccine-derived polioviruses (VDPVs) for an extended period, which could pose a major threat to polio eradication programs. Since 1995, sixteen VDPV populations have been isolated from 14 patients with immunodeficiency in Iran. For these patients, vaccine-associated paralysis, mostly in >1 extremity, was the first manifestation of primary immunodeficiency. Seven patients with humoral immunodeficiency cleared VDPV infection more frequently than did 6 patients with combined immunodeficiencies. Our results raise questions about manifestations of VDPVs in immunodeficient patients and the role of cellular immunity against enterovirus infections. On the basis of an association between VDPVs and immunodeficiency, we advocate screening of patients with primary immunodeficiency for shedding of polioviruses. PMID:27648512

  2. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    PubMed Central

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  3. Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

    SciTech Connect

    Sonenberg, N.; Guertin, D.; Lee, K.A.W.

    1982-12-01

    Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

  4. A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

    Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells. PMID:7666528

  5. Update on vaccine-derived polioviruses - worldwide, July 2012-December 2013.

    PubMed

    Diop, Ousmane M; Burns, Cara C; Wassilak, Steven G; Kew, Olen M

    2014-03-21

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide. One of the main tools used in polio eradication efforts has been live, attenuated oral poliovirus vaccine (OPV), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but then it provides long-term protection against paralytic disease through durable humoral immunity. Rare cases of vaccine-associated paralytic poliomyelitis can occur among immunologically normal OPV recipients, their contacts, and persons who are immunodeficient. In addition, vaccine-derived polioviruses (VDPVs) can emerge in areas with low OPV coverage to cause polio outbreaks and can replicate for years in persons who have primary, B-cell immunodeficiencies. This report updates previous surveillance summaries and describes VDPVs detected worldwide during July 2012-December 2013. Those include a new circulating VDPV (cVDPV) outbreak identified in Pakistan in 2012, with spread to Afghanistan; an outbreak in Afghanistan previously identified in 2009 that continued into 2013; a new outbreak in Chad that spread to Cameroon, Niger, and northeastern Nigeria; and an outbreak that began in Somalia in 2008 that continued and spread to Kenya in 2013. A large outbreak in Nigeria that was identified in 2005 was nearly stopped by the end of 2013. Additionally, 10 newly identified persons in eight countries were found to excrete immunodeficiency-associated VDPVs (iVDPVs), and VDPVs were found among immunocompetent persons and environmental samples in 13 countries. Because the majority of VDPV isolates are type 2, the World Health Organization has developed a plan for coordinated worldwide replacement of trivalent OPV (tOPV) with bivalent OPV (bOPV; types 1 and 3) by 2016, preceded by introduction of at least 1 dose of inactivated poliovirus vaccine (IPV) containing all three poliovirus serotypes into routine immunization schedules worldwide to ensure high population

  6. Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

    PubMed

    Feduchi, E; Aldabe, R; Novoa, I; Carrasco, L

    1995-12-15

    The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

  7. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

    PubMed Central

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

    2016-01-01

    ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

  8. The role of older children and adults in wild poliovirus transmission

    PubMed Central

    Blake, Isobel M.; Martin, Rebecca; Goel, Ajay; Khetsuriani, Nino; Everts, Johannes; Wolff, Christopher; Wassilak, Steven; Aylward, R. Bruce; Grassly, Nicholas C.

    2014-01-01

    As polio eradication inches closer, the absence of poliovirus circulation in most of the world and imperfect vaccination coverage are resulting in immunity gaps and polio outbreaks affecting adults. Furthermore, imperfect, waning intestinal immunity among older children and adults permits reinfection and poliovirus shedding, prompting calls to extend the age range of vaccination campaigns even in the absence of cases in these age groups. The success of such a strategy depends on the contribution to poliovirus transmission by older ages, which has not previously been estimated. We fit a mathematical model of poliovirus transmission to time series data from two large outbreaks that affected adults (Tajikistan 2010, Republic of Congo 2010) using maximum-likelihood estimation based on iterated particle-filtering methods. In Tajikistan, the contribution of unvaccinated older children and adults to transmission was minimal despite a significant number of cases in these age groups [reproduction number, R = 0.46 (95% confidence interval, 0.42–0.52) for >5-y-olds compared to 2.18 (2.06–2.45) for 0- to 5-y-olds]. In contrast, in the Republic of Congo, the contribution of older children and adults was significant [R = 1.85 (1.83–4.00)], perhaps reflecting sanitary and socioeconomic variables favoring efficient virus transmission. In neither setting was there evidence for a significant role of imperfect intestinal immunity in the transmission of poliovirus. Bringing the immunization response to the Tajikistan outbreak forward by 2 wk would have prevented an additional 130 cases (21%), highlighting the importance of early outbreak detection and response. PMID:25002465

  9. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  10. Interferons and Ribavirin Effectively Inhibit Norwalk Virus Replication in Replicon-Bearing Cells▿

    PubMed Central

    Chang, Kyeong-Ok; George, David W.

    2007-01-01

    The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-α) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-γ and ribavirin for their effects on NV replication in the cells. Like IFN-α, IFN-γ inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED50) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED50 calculated as approximately 40 μM. The combination of IFN-α and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 μM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis. PMID:17855555

  11. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout.

    PubMed

    Gläsker, Sabine; Lulla, Aleksei; Lulla, Valeria; Couderc, Therese; Drexler, Jan Felix; Liljeström, Peter; Lecuit, Marc; Drosten, Christian; Merits, Andres; Kümmerer, Beate Mareike

    2013-07-15

    Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. A VRP based CHIKV neutralization assay using Gluc as readout represents a fast and useful method to

  12. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia.

    PubMed

    Althabegoiti, María Julia; Ormeño-Orrillo, Ernesto; Lozano, Luis; Torres Tejerizo, Gonzalo; Rogel, Marco Antonio; Mora, Jaime; Martínez-Romero, Esperanza

    2014-01-08

    Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

  13. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

    PubMed Central

    2014-01-01

    Background Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization

  14. Antibody to a synthetic nonapeptide corresponding to the NH2 terminus of poliovirus genome-linked protein VPg reacts with native VPg and inhibits in vitro replication of poliovirus RNA.

    PubMed Central

    Morrow, C D; Dasgupta, A

    1983-01-01

    A synthetic nonapeptide corresponding to the N-terminal sequence of poliovirus genome-linked protein (VPg) was linked to bovine serum albumin and used to raise antibodies in rabbits. The antipeptide antibodies specifically precipitated the nonapeptide, native VPg, and VPg-linked poliovirion RNA. The antipeptide antibodies inhibited host factor-stimulated, poliovirus replicase-catalyzed in vitro synthesis of full-length (35S) RNA in response to virion RNA. Oligouridylic acid-stimulated RNA synthesis was not affected by the antipeptide antibodies. Preincubation of the antibodies with excess nonapeptide reversed the antipeptide antibody-mediated inhibition of host factor-stimulated RNA synthesis by the poliovirus replicase. A role for VPg in the in vitro replication of poliovirus RNA genome is discussed. Images PMID:6312101

  15. Evaluation of neurovirulence and biodistribution of Venezuelan equine encephalitis replicon particles expressing herpes simplex virus type 2 glycoprotein D.

    PubMed

    Kowalski, Jacek; Adkins, Karissa; Gangolli, Seema; Ren, Jian; Arendt, Heather; DeStefano, Joanne; Obregon, Jennifer; Tummolo, Donna; Natuk, Robert J; Brown, Tom P; Parks, Christopher L; Udem, Stephen A; Long, Deborah

    2007-03-08

    The safety of a propagation-defective Venezuelan equine encephalitis virus (VEEV) replicon particle vaccine was examined in mice. After intracranial inoculation we observed approximately 5% body weight loss, modest inflammatory changes in the brain, genome replication, and foreign gene expression. These changes were transient and significantly less severe than those caused by TC-83, a live-attenuated vaccinal strain of VEEV that has been safely used to immunize military personnel and laboratory workers. Replicon particles injected intramuscularly or intravenously were detected at limited sites 3 days post-administration, and were undetectable by day 22. There was no evidence of dissemination to spinal cord or brain after systemic administration. These results demonstrate that propagation-defective VEEV replicon particles are minimally neurovirulent and lack neuroinvasive potential.

  16. Kunjin virus replicon-based vaccines expressing Ebola virus glycoprotein GP protect the guinea pig against lethal Ebola virus infection.

    PubMed

    Reynard, O; Mokhonov, V; Mokhonova, E; Leung, J; Page, A; Mateo, M; Pyankova, O; Georges-Courbot, M C; Raoul, H; Khromykh, A A; Volchkov, V E

    2011-11-01

    Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)-derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor-truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection.

  17. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

    USDA-ARS?s Scientific Manuscript database

    Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating...

  18. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

    PubMed

    Pohjala, Leena; Utt, Age; Varjak, Margus; Lulla, Aleksei; Merits, Andres; Ahola, Tero; Tammela, Päivi

    2011-01-01

    Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti

  19. Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV.

    PubMed

    Traxler, G S; Anderson, E; LaPatra, S E; Richard, J; Shewmaker, B; Kurath, G

    1999-11-30

    A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.

  20. Rapid micromotor-based naked-eye immunoassay.

    PubMed

    de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

    2017-05-15

    A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H2O2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL(-1) in just 2min, using ultrasmall (50µL) sample volumes.

  1. Daylight Observations of Venus with Naked Eye in the Goryeosa

    NASA Astrophysics Data System (ADS)

    Lee, Ki-Won

    2017-03-01

    In this paper, we investigate the observations of Venus in daytime that are recorded in the Goryeosa (History of the Goryeo Dynasty, A.D. 918-1392). There are a total of 167 accounts of such observations in this historical book, spanning a period of 378 yr (from 1014 to 1392). These include six accounts where the days of the observation are not specified and two accounts where the phase angles are outside the calculation range of the equation used in our study. We analyze the number distribution of 164 accounts in 16 yr intervals covering the period from 1023 to 1391. We find that this distribution shows its minimum at around 1232, when the Goryeo dynasty moved the capital to the Ganghwa Island because of the Mongol invasion, and its maximum at around 1390, about the time when the dynasty fell. In addition, we calculate the azimuth, altitude, solar elongation, and apparent magnitude of Venus at sunset for 159 observations, excluding the eight accounts mentioned above, using the DE 406 ephemeris and modern astronomical algorithms. We find that the average elongation and magnitude of Venus on the days of those accounts were and -4.5, respectively, whereas the minimum magnitude was -3.8. The results of this study are useful for estimating the practical conditions for observing Venus in daylight with the naked eye and they also provide additional insight into the corresponding historical accounts contained in the Goryeosa.

  2. TORTORA discovery of Naked-Eye Burst fast optical variability

    NASA Astrophysics Data System (ADS)

    Beskin, Grigory; Karpov, Sergey; Bondar, Sergey; Greco, Giuseppe; Guarnieri, Adriano; Bartolini, Corrado; Piccioni, Adalberto; Molinari, Emilio; Chincarini, Guido

    2008-10-01

    Features characterizing gamma-ray bursts in the different spectral bands may be a clue for the nature of their inner engine. Up to now, only several bursts have been observed in optical band during the gamma activity, and the only one-GRB080319B-was covered from rise till fall with high temporal resolution. Here we discuss these data, acquired with TORTORA fast wide-field monitoring optical camera, as well as results of its analysis. The camera observed the position of Naked-Eye Burst, GRB080318B, before, during and after the trigger. It detected the fast rise of optical emission, which reached the peak of V 5.3 at the eighteenth second, had a complex evolution till T+43s and monotonously faded then. The brightest part of the light curve contains two 15-20 s segments with different fluxes, each having two clearly-seen peaks of 5-8 s duration; all four peaks look quasi-periodic with separation of 9 s. There is no clear evidence of any sub-second variability. However, there are signs of quasi-periodic variability on 1s time scale at around the last peak (T+40 till T+50). The general properties of the optical light curve and its variability time scales look similar to the gamma one, but there is no clear correlation between them. This raises serious problems in interpretation of mechanisms generating such variability.

  3. Seeing Beyond the Naked Eye in a Planetarium

    NASA Astrophysics Data System (ADS)

    Fairall, A.

    2005-12-01

    I have a philosophy that the traditional naked-eye sky, as usually shown in planetariums, should only be an introductory step in portraying the Universe. Consequently, over the years I have produced 'inter alia' various versions of an enhanced Milky Way (the latest based on Axel Mellenger's panorama), the extragalactic sky and the radio sky for projection on planetarium domes. I also put together a three-dimensional planetarium show-the audience being equipped with ChromDepth(tm) spectacles- which stepped from the Solar System to the cosmic microwave background. The advent of digital technology now makes all this much easier. Currently, Labyrinth, a visualization program developed in-house, serves much the same function as the Hayden Planetarium's Partiview, but also permits rendering and fl y-throughs of large-scale structures. It allows viewers to explore local cosmography. Labyrinth can produce images that operate with the 3-D spectacles; we have also produced a version of Partiview that does the same.

  4. Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat.

    PubMed

    Park, Thomas J; Reznick, Jane; Peterson, Bethany L; Blass, Gregory; Omerbašić, Damir; Bennett, Nigel C; Kuich, P Henning J L; Zasada, Christin; Browe, Brigitte M; Hamann, Wiebke; Applegate, Daniel T; Radke, Michael H; Kosten, Tetiana; Lutermann, Heike; Gavaghan, Victoria; Eigenbrod, Ole; Bégay, Valérie; Amoroso, Vince G; Govind, Vidya; Minshall, Richard D; Smith, Ewan St J; Larson, John; Gotthardt, Michael; Kempa, Stefan; Lewin, Gary R

    2017-04-21

    The African naked mole-rat's (Heterocephalus glaber) social and subterranean lifestyle generates a hypoxic niche. Under experimental conditions, naked mole-rats tolerate hours of extreme hypoxia and survive 18 minutes of total oxygen deprivation (anoxia) without apparent injury. During anoxia, the naked mole-rat switches to anaerobic metabolism fueled by fructose, which is actively accumulated and metabolized to lactate in the brain. Global expression of the GLUT5 fructose transporter and high levels of ketohexokinase were identified as molecular signatures of fructose metabolism. Fructose-driven glycolytic respiration in naked mole-rat tissues avoids feedback inhibition of glycolysis via phosphofructokinase, supporting viability. The metabolic rewiring of glycolysis can circumvent the normally lethal effects of oxygen deprivation, a mechanism that could be harnessed to minimize hypoxic damage in human disease. Copyright © 2017, American Association for the Advancement of Science.

  5. Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

    PubMed

    Yu, Yun-Zhou; Li, Na; Wang, Wen-Bin; Wang, Shuang; Ma, Yao; Yu, Wei-Yuan; Sun, Zhi-Wei

    2010-11-03

    Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.

  6. Lack of Interference with Immunogenicity of a Chimeric Alphavirus Replicon Particle-Based Influenza Vaccine by Preexisting Antivector Immunity

    PubMed Central

    Vajdy, Michael; Lian, Ying; Perri, Silvia; Greer, Catherine E.; Legg, Harold S.; Galli, Grazia; Saletti, Giulietta; Otten, Gillis R.; Rappuoli, Rino; Barnett, Susan W.; Polo, John M.

    2012-01-01

    Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens. PMID:22623651

  7. Utilization of Two Distinct Modes of Replication by a Hybrid Plasmid Constructed In Vitro from Separate Replicons

    PubMed Central

    Timmis, Kenneth; Cabello, Felipe; Cohen, Stanley N.

    1974-01-01

    A hybrid plasmid, pSC134, that codes for two distinct sets of replication functions has been constructed in vitro by ligation of EcoRI endonuclease-cleaved pSC101 and Col E1 plasmid replicons, and has been introduced into Escherichia coli by transformation. The replication properties of the pSC134 plasmid in DNA polymerase I-defective mutants or in the presence of chloramphenicol indicate that this hybrid plasmid can utilize the functionally distinct modes of replication specified by both of its parent replicons. Images PMID:4612523

  8. Improved thermal stability of oxide-supported naked gold nanoparticles by ligand-assisted pinning

    SciTech Connect

    Moreno, C; Divins, N. J.; Gazquez, Jaume; Varela, Maria; Angurell, I; Llorca, J

    2012-01-01

    We report a method to improve the thermal stability, up to 900 C, of bare-metal (naked) gold nanoparticles supported on top of SiO{sub 2} and SrTiO{sub 3} substrates via ligand-assisted pinning. This approach leads to monodisperse naked gold nanoparticles without significant sintering after thermal annealing in air at 900 C. The ligand-assisted pinning mechanism is described.

  9. Genome sequencing reveals insights into physiology and longevity of the naked mole rat.

    PubMed

    Kim, Eun Bae; Fang, Xiaodong; Fushan, Alexey A; Huang, Zhiyong; Lobanov, Alexei V; Han, Lijuan; Marino, Stefano M; Sun, Xiaoqing; Turanov, Anton A; Yang, Pengcheng; Yim, Sun Hee; Zhao, Xiang; Kasaikina, Marina V; Stoletzki, Nina; Peng, Chunfang; Polak, Paz; Xiong, Zhiqiang; Kiezun, Adam; Zhu, Yabing; Chen, Yuanxin; Kryukov, Gregory V; Zhang, Qiang; Peshkin, Leonid; Yang, Lan; Bronson, Roderick T; Buffenstein, Rochelle; Wang, Bo; Han, Changlei; Li, Qiye; Chen, Li; Zhao, Wei; Sunyaev, Shamil R; Park, Thomas J; Zhang, Guojie; Wang, Jun; Gladyshev, Vadim N

    2011-10-12

    The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

  10. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat.

    PubMed

    Seluanov, Andrei; Hine, Christopher; Azpurua, Jorge; Feigenson, Marina; Bozzella, Michael; Mao, Zhiyong; Catania, Kenneth C; Gorbunova, Vera

    2009-11-17

    The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed "early contact inhibition." Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27(Kip1). In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16(Ink4a). Furthermore, we show that the roles of p16(Ink4a) and p27(Kip1) in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16(Ink4a), and regular contact inhibition is controlled by p27(Kip1). We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat.

  11. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat

    PubMed Central

    Seluanov, Andrei; Hine, Christopher; Azpurua, Jorge; Feigenson, Marina; Bozzella, Michael; Mao, Zhiyong; Catania, Kenneth C.; Gorbunova, Vera

    2009-01-01

    The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed “early contact inhibition.” Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27Kip1. In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16Ink4a. Furthermore, we show that the roles of p16Ink4a and p27Kip1 in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16Ink4a, and regular contact inhibition is controlled by p27Kip1. We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat. PMID:19858485

  12. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.

    PubMed

    Date, Tomoko; Kato, Takanobu; Miyamoto, Michiko; Zhao, Zijiang; Yasui, Kotaro; Mizokami, Masashi; Wakita, Takaji

    2004-05-21

    A hepatitis C virus genotype 2a subgenomic replicon, JFH-1 replicon, was previously established using the consensus sequence of clone JFH-1 from a patient with fulminant hepatitis and, in a previous report, was indicated to replicate efficiently in Huh7. Here the replication of JFH-1 replicon was tested in HepG2, a human hepatocyte-derived cell line, and in IMY-N9, a cell line developed by fusing human hepatocytes and HepG2 cells. Following transfection with in vitro transcribed replicon RNA and selection by cultivation with G418, colonies formed in both cell lines although at efficiencies substantially lower than those of Huh7. The H2476L mutation identified in the Huh7 replicon in our previous study increased the colony formation efficiencies of the JFH-1 replicon in HepG2 and IMY-N9 cells. Higher amounts of replicon RNA were detected in IMY-N9 clones than in HepG2 clones by real time detection reverse transcription-PCR, and replicon RNA replication and viral protein expression were confirmed by Northern and Western blotting in isolated clones. Sequencing of replicon RNAs revealed that mutations found in hepatitis C virus-derived regions were not identical and that two of nine HepG2 clones and three of nine IMY-N9 clones had no or one synonymous mutation. This system with the JFH-1 replicon and three cell lines is useful not only for estimating the cellular factors affecting viral activity but also for clarifying the common gene response of the host.

  13. Blunted neuronal calcium response to hypoxia in naked mole-rat hippocampus.

    PubMed

    Peterson, Bethany L; Larson, John; Buffenstein, Rochelle; Park, Thomas J; Fall, Christopher P

    2012-01-01

    Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals.

  14. Blunted Neuronal Calcium Response to Hypoxia in Naked Mole-Rat Hippocampus

    PubMed Central

    Peterson, Bethany L.; Larson, John; Buffenstein, Rochelle

    2012-01-01

    Naked mole-rats are highly social and strictly subterranean rodents that live in large communal colonies in sealed and chronically oxygen-depleted burrows. Brain slices from naked mole-rats show extreme tolerance to hypoxia compared to slices from other mammals, as indicated by maintenance of synaptic transmission under more hypoxic conditions and three fold longer latency to anoxic depolarization. A key factor in determining whether or not the cellular response to hypoxia is reversible or leads to cell death may be the elevation of intracellular calcium concentration. In the present study, we used fluorescent imaging techniques to measure relative intracellular calcium changes in CA1 pyramidal cells of hippocampal slices during hypoxia. We found that calcium accumulation during hypoxia was significantly and substantially attenuated in slices from naked mole-rats compared to slices from laboratory mice. This was the case for both neonatal (postnatal day 6) and older (postnatal day 20) age groups. Furthermore, while both species demonstrated more calcium accumulation at older ages, the older naked mole-rats showed a smaller calcium accumulation response than even the younger mice. A blunted intracellular calcium response to hypoxia may contribute to the extreme hypoxia tolerance of naked mole-rat neurons. The results are discussed in terms of a general hypothesis that a very prolonged or arrested developmental process may allow adult naked mole-rat brain to retain the hypoxia tolerance normally only seen in neonatal mammals. PMID:22363676

  15. Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

    PubMed

    Kamrud, Kurt I; Alterson, Kim D; Andrews, Chasity; Copp, Laura O; Lewis, Whitney C; Hubby, Bolyn; Patel, Deepa; Rayner, Jonathan O; Talarico, Todd; Smith, Jonathan F

    2008-07-16

    The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

  16. Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant alphavirus replicon particles.

    PubMed

    Goldberg, Stacie M; Bartido, Shirley M; Gardner, Jason P; Guevara-Patiño, José A; Montgomery, Stephanie C; Perales, Miguel-Angel; Maughan, Maureen F; Dempsey, JoAnn; Donovan, Gerald P; Olson, William C; Houghton, Alan N; Wolchok, Jedd D

    2005-11-15

    Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen. Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection. T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone. Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.

  17. Effect of buffer on the immune response to trivalent oral poliovirus vaccine in Bangladesh: a community based randomized controlled trial.

    PubMed

    Chandir, Subhash; Ahamed, Kabir U; Baqui, Abdullah H; Sutter, Roland W; Okayasu, Hiromasa; Pallansch, Mark A; Oberste, Mark S; Moulton, Lawrence H; Halsey, Neal A

    2014-11-01

    Polio eradication efforts have been hampered by low responses to trivalent oral poliovirus vaccine (tOPV) in some developing countries. Since stomach acidity may neutralize vaccine viruses, we assessed whether administration of a buffer solution could improve the immunogenicity of tOPV. Healthy infants 4-6 weeks old in Sylhet, Bangladesh, were randomized to receive tOPV with or without a sodium bicarbonate and sodium citrate buffer at age 6, 10, and 14 weeks. Levels of serum neutralizing antibodies for poliovirus types 1, 2, and 3 were measured before and after vaccination, at 6 and 18 weeks of age, respectively. Serologic response rates following 3 doses of tOPV for buffer recipients and control infants were 95% and 88% (P=.065), respectively, for type 1 poliovirus; 95% and 97% (P=.543), respectively, for type 2 poliovirus; and 90% and 89% (P=.79), respectively, for type 3 poliovirus. Administration of a buffer solution prior to vaccination was not associated with statistically significant increases in the immune response to tOPV; however, a marginal 7% increase (P=.065) in serologic response to poliovirus type 1 was observed. NCT01579825. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn sup 2+

    SciTech Connect

    Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G. )

    1989-09-01

    The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg{sup 2+}. In this paper, the effect of Zn{sup 2+} on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg{sup 2+}. In the presence of Zn{sup 2+}, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn{sup 2+}. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn{sup 2+}. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.

  19. Changes in population dynamics during long-term evolution of sabin type 1 poliovirus in an immunodeficient patient.

    PubMed

    Odoom, John K; Yunus, Zaira; Dunn, Glynis; Minor, Philip D; Martín, Javier

    2008-09-01

    The evolution of the Sabin strain of type 1 poliovirus in a hypogammaglobulinemia patient for a period of 649 days is described. Twelve poliovirus isolates from sequential stool samples encompassing days 21 to 649 after vaccination with Sabin 1 were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin 1 strain. Poliovirus isolates from the immunodeficient patient evolved gradually toward non-temperature-sensitive and neurovirulent phenotypes, accumulating mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. Analysis of plaque-purified viruses from stool samples revealed complex genetic and evolutionary relationships between the poliovirus strains. The generation of various coevolving genetic lineages incorporating different mutations was observed at early stages of virus excretion. The main driving force for genetic diversity appeared to be the selection of mutations at attenuation sites, particularly in the 5' noncoding region and the VP1 BC loop. Recombination between virus strains from the two main lineages was observed between days 63 and 88. Genetic heterogeneity among plaque-purified viruses at each time point seemed to decrease with time, and only viruses belonging to a unique genotypic lineage were seen from day 105 after vaccination. The relevance of vaccine-derived poliovirus strains for disease surveillance and future polio immunization policies is discussed in the context of the Global Polio Eradication Initiative.

  20. Changes in Population Dynamics during Long-Term Evolution of Sabin Type 1 Poliovirus in an Immunodeficient Patient▿

    PubMed Central

    Odoom, John K.; Yunus, Zaira; Dunn, Glynis; Minor, Philip D.; Martín, Javier

    2008-01-01

    The evolution of the Sabin strain of type 1 poliovirus in a hypogammaglobulinemia patient for a period of 649 days is described. Twelve poliovirus isolates from sequential stool samples encompassing days 21 to 649 after vaccination with Sabin 1 were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin 1 strain. Poliovirus isolates from the immunodeficient patient evolved gradually toward non-temperature-sensitive and neurovirulent phenotypes, accumulating mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. Analysis of plaque-purified viruses from stool samples revealed complex genetic and evolutionary relationships between the poliovirus strains. The generation of various coevolving genetic lineages incorporating different mutations was observed at early stages of virus excretion. The main driving force for genetic diversity appeared to be the selection of mutations at attenuation sites, particularly in the 5′ noncoding region and the VP1 BC loop. Recombination between virus strains from the two main lineages was observed between days 63 and 88. Genetic heterogeneity among plaque-purified viruses at each time point seemed to decrease with time, and only viruses belonging to a unique genotypic lineage were seen from day 105 after vaccination. The relevance of vaccine-derived poliovirus strains for disease surveillance and future polio immunization policies is discussed in the context of the Global Polio Eradication Initiative. PMID:18596089

  1. Prolonged Excretion of Poliovirus among Individuals with Primary Immunodeficiency Disorder: An Analysis of the World Health Organization Registry.

    PubMed

    Macklin, Grace; Liao, Yi; Takane, Marina; Dooling, Kathleen; Gilmour, Stuart; Mach, Ondrej; Kew, Olen M; Sutter, Roland W

    2017-01-01

    Individuals with primary immunodeficiency disorder may excrete poliovirus for extended periods and will constitute the only remaining reservoir of virus after eradication and withdrawal of oral poliovirus vaccine. Here, we analyzed the epidemiology of prolonged and chronic immunodeficiency-related vaccine-derived poliovirus cases in a registry maintained by the World Health Organization, to identify risk factors and determine the length of excretion. Between 1962 and 2016, there were 101 cases, with 94/101 (93%) prolonged excretors and 7/101 (7%) chronic excretors. We documented an increase in incidence in recent decades, with a shift toward middle-income countries, and a predominance of poliovirus type 2 in 73/101 (72%) cases. The median length of excretion was 1.3 years (95% confidence interval: 1.0, 1.4) and 90% of individuals stopped excreting after 3.7 years. Common variable immunodeficiency syndrome and residence in high-income countries were risk factors for long-term excretion. The changing epidemiology of cases, manifested by the greater incidence in recent decades and a shift to from high- to middle-income countries, highlights the expanding risk of poliovirus transmission after oral poliovirus vaccine cessation. To better quantify and reduce this risk, more sensitive surveillance and effective antiviral therapies are needed.

  2. Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland.

    PubMed Central

    Pöyry, T; Stenvik, M; Hovi, T

    1988-01-01

    During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign. PMID:2833160

  3. Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

    PubMed

    Strauss, Mike; Filman, David J; Belnap, David M; Cheng, Naiqian; Noel, Roane T; Hogle, James M

    2015-04-01

    Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin

  4. Development of inactivated poliovirus vaccine from Sabin strains: A progress report.

    PubMed

    Okayasu, Hiromasa; Sein, Carolyn; Hamidi, Ahd; Bakker, Wilfried A M; Sutter, Roland W

    2016-11-01

    The Global Polio Eradication Initiative (GPEI) has seen significant progress since it began in 1988, largely due to the worldwide use of oral poliovirus vaccine (OPV). In order to achieve polio eradication the global cessation of OPV is necessary because OPV contains live attenuated poliovirus, which in rare circumstances could re-gain wild poliovirus (WPV) characteristics with potential to establish transmission. The GPEI endgame strategy for the period 2013-2018 recommends the globally synchronised sequential cessation of the Sabin strains contained in the OPV, starting with type 2 Sabin. The withdrawal of Sabin type 2 took place in April 2016, with the introduction of at least one dose of inactivated poliovirus vaccine (IPV) as a risk mitigation strategy. The introduction of IPV into 126 countries since 2013 has required a rapid scale-up of IPV production by the two manufacturers supplying the global public sector market. This scale-up has been fraught with challenges, resulting in reductions of 40-50% of initial supply commitments. Consequently, 22 countries will not be supplied until 2018, and another 23 countries will experience serious stock-outs. In the last decade repeated calls-for-action were made to the global community to invigorate their vision and investment in developing "new poliovirus vaccines" including the development of IPV from less-virulent strains, such as Sabin-IPV (S-IPV). The conventional Salk-IPV production is limited to high-income industrialized-country manufacturers due to the containment requirements (i.e., high sanitation, low force-of-poliovirus-infection, and high population immunity). The use of Sabin strains in the production of S-IPV carries a lower biosafety risk, and was determined to be suitable for production in developing countries, expanding the manufacturing base and making IPV more affordable and accessible in the long term. Significant progress in the S-IPV has been made since 2006. S-IPV is now licensed as S-IPV in

  5. A naked-eye based strategy for semiquantitative immunochromatographic assay.

    PubMed

    Zhang, Daohong; Li, Peiwu; Zhang, Qi; Li, Ran; Zhang, Wen; Ding, Xiaoxia; Li, Chang Ming

    2012-08-31

    It is critical to develop a cost-effective quantitative/semiquantitative assay for rapid diagnosis and on-site detection of toxic or harmful substances. Here, a naked-eye based semiquantitative immunochromatographic strip (NSI-strip) was developed, on which three test lines (TLs, TL-I, TL-II and TL-III) were dispensed on a nitrocellulose membrane to form the test zone. Similar as the traditional strip assay for small molecule, the NSI-strip assay was also based on the competitive theory, difference was that the analyte competed three times with the capture reagent for the limited number of antibody binding sites. After the assay, the number of TLs developed in the test zone was inversely proportional to the analyte concentration, thus analyte content levels could be determined by observing the appeared number of TLs. Taking aflatoxin B(1) as the model analyte, visual detection limit of the NSI-strip was 0.06 ng mL(-1) and threshold concentrations for TL-I-III were 0.125, 0.5, and 2.0 ng mL(-1), respectively. Therefore, according to the appeared number of TLs, the following concentration ranges would be detectable by visual examination: 0-0.06 ng mL(-1) (negative samples), and 0.06-0.125 ng mL(-1), 0.125-0.5 ng mL(-1), 0.5-2.0 ng mL(-1) and >2.0 ng mL(-1) (positive samples). That was to say, compared to traditional strips the NSI-strip could offer more parameter information of the target analyte content. In this way, the NSI-strip improved the qualitative presence/absence detection of traditional strips by measuring the content (range) of target analytes semiquantitatively.

  6. Seeing protein monolayers with naked eye through plasmonic Fano resonances.

    PubMed

    Yanik, Ahmet A; Cetin, Arif E; Huang, Min; Artar, Alp; Mousavi, S Hossein; Khanikaev, Alexander; Connor, John H; Shvets, Gennady; Altug, Hatice

    2011-07-19

    We introduce an ultrasensitive label-free detection technique based on asymmetric Fano resonances in plasmonic nanoholes with far reaching implications for point-of-care diagnostics. By exploiting extraordinary light transmission phenomena through high-quality factor (Q(solution) ∼ 200) subradiant dark modes, we experimentally demonstrate record high figures of merits (FOMs as high as 162) for intrinsic detection limits surpassing that of the gold standard prism coupled surface-plasmon sensors (Kretschmann configuration). Our experimental record high sensitivities are attributed to the nearly complete suppression of the radiative losses that are made possible by the high structural quality of the fabricated devices as well as the subradiant nature of the resonances. Steep dispersion of the plasmonic Fano resonance profiles in high-quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances and the associated Wood's anomalies. To fabricate high optical-quality sensors, we introduce a high-throughput lift-off free evaporation fabrication technique with extremely uniform and precisely controlled nanofeatures over large areas, leading to resonance line-widths comparable to that of the ideally uniform structures as confirmed by our time-domain simulations. The demonstrated label-free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation.

  7. Seeing protein monolayers with naked eye through plasmonic Fano resonances

    PubMed Central

    Yanik, Ahmet A.; Cetin, Arif E.; Huang, Min; Artar, Alp; Mousavi, S. Hossein; Khanikaev, Alexander; Connor, John H.; Shvets, Gennady; Altug, Hatice

    2011-01-01

    We introduce an ultrasensitive label-free detection technique based on asymmetric Fano resonances in plasmonic nanoholes with far reaching implications for point-of-care diagnostics. By exploiting extraordinary light transmission phenomena through high-quality factor (Qsolution ∼ 200) subradiant dark modes, we experimentally demonstrate record high figures of merits (FOMs as high as 162) for intrinsic detection limits surpassing that of the gold standard prism coupled surface-plasmon sensors (Kretschmann configuration). Our experimental record high sensitivities are attributed to the nearly complete suppression of the radiative losses that are made possible by the high structural quality of the fabricated devices as well as the subradiant nature of the resonances. Steep dispersion of the plasmonic Fano resonance profiles in high-quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances and the associated Wood’s anomalies. To fabricate high optical-quality sensors, we introduce a high-throughput lift-off free evaporation fabrication technique with extremely uniform and precisely controlled nanofeatures over large areas, leading to resonance line-widths comparable to that of the ideally uniform structures as confirmed by our time-domain simulations. The demonstrated label-free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation. PMID:21715661

  8. Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics.

    PubMed

    Vlaicu, Ovidiu; Selescu, Tudor; Pastrama, Florin; Munteanu, Cristian; Riva, Laura; Dubuisson, Jean; Rouille, Yves; Popescu, Costin-Ioan

    2017-08-01

    Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.

    PubMed

    Pushko, P; Bray, M; Ludwig, G V; Parker, M; Schmaljohn, A; Sanchez, A; Jahrling, P B; Smith, J F

    2000-08-15

    RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

  10. A polyprotein-expressing salmonid alphavirus replicon induces modest protection in atlantic salmon (Salmo salar) against infectious pancreatic necrosis.

    PubMed

    Abdullah, Azila; Olsen, Christel M; Hodneland, Kjartan; Rimstad, Espen

    2015-01-19

    Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

  11. Virus removal during groundwater recharge: effects of infiltration rate on adsorption of poliovirus to soil

    SciTech Connect

    Vaughn, J.M.; Landry, E.F.; Beckwith, C.A.; Thomas, M.Z.

    1981-01-01

    Studies were conducted to determine the influence of infiltration rate on poliovirus removal during groundwater recharge with tertiary-treated wastewater effluents. Experiments were conducted at a uniquely designed, field-situated test recharge basin facility through which some 62,000 m/sup 3/ of sewage had been previously applied. Recharge at high infiltration rates (75 to 100 cm/h) resulted in the movement of considerable numbers of seeded poliovirus to the groundwater. Moderately reduced infiltration rates (6 cm/h) affected significantly improved virus removal. Very low infiltration rates (0.5 to 1.0 cm/h), achieved by partial clogging of the test basin, yielded the greatest virus removal efficiencies.

  12. Characteristics of districts in Pakistan with persistent transmission of wild poliovirus, 2000-2001.

    PubMed

    Lowther, S A; Mir, T; Bile, M K; Abdul Hafiz, R; Mounts, A W

    2004-01-01

    We sought to identify factors associated with being a reservoir district for wild poliovirus in Pakistan. Differences between reservoir and non-reservoir districts were identified using acute flaccid paralysis surveillance data, population census statistics and data from a survey of district health officials (DHOs). Of the 11 poliovirus reservoir districts identified, population density was significantly higher (median 550 persons/km2) than the non-reservoirs (median 175 persons/km2). DHOs from reservoir districts more often reported that planning was affected by refugees and they had more frequent DHO transfers compared with non-reservoir districts. Multivariate analysis confirmed that reservoirs more often had high population density and frequent DHO transfers. Assessment of district-level and management characteristics can supplement surveillance methods to further improve health programmes.

  13. Naked oats: metabolisable energy yield from a range of varieties in broilers, cockerels and turkeys.

    PubMed

    MacLeod, M G; Valentine, J; Cowan, A; Wade, A; McNeill, L; Bernard, K

    2008-05-01

    1. Naked oats belong to the same species as 'common oats', Avena sativa, but have a non-lignified husk which readily becomes detached during harvesting. The absence of the indigestible husk can be predicted to give an increased metabolisable energy (ME) content for poultry. 2. Measurements of true metabolisable energy (TME(N)) were performed on 3-week-old broiler chicks (Cobb males), adult cockerels (ISA Brown, greater than one year old) and 7-week-old turkeys (BUT T8 males). The measurements were repeated in 2000, 2001 and 2002, with some measurements on a subset of varieties in 2004. 3. High-oil naked oat lines yielded 12% more energy (TME(N)) than wheat. Naked oats, excluding the experimental high-oil lines, yielded 8.5% more energy than simultaneously assayed wheat samples. 4. In samples from the 2004 harvest, conventional oats gave TME(N) values about 13% lower than those of wheat. 5. The addition of beta-glucanase produced an increase of about 4% in the apparent metabolisable energy (AME) of oats for broiler chickens. This effect was associated with a 70% decrease in the jejunal viscosity of broilers receiving a 500 g/kg naked-oat diet. 6. The oil content of naked oats was about 5 times greater than that of wheat, with the high-oil lines rising to more than 6 times greater. Naked oats had a lower starch content than wheat but not sufficiently lower to negate the energy benefits of the higher oil content. The crude protein (CP) contents of naked oats were similar to those of wheat, with the high-oil varieties tending to be higher in CP also.

  14. Combined Alphavirus Replicon Particle Vaccine Induces Durable and Cross-Protective Immune Responses against Equine Encephalitis Viruses

    PubMed Central

    Glass, Pamela J.; Bakken, Russell R.; Barth, James F.; Lind, Cathleen M.; da Silva, Luis; Hart, Mary Kate; Rayner, Jonathan; Alterson, Kim; Custer, Max; Dudek, Jeanne; Owens, Gary; Kamrud, Kurt I.; Parker, Michael D.; Smith, Jonathan

    2014-01-01

    ABSTRACT Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal. IMPORTANCE Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the

  15. Long-term excretion of vaccine-derived poliovirus by a healthy child.

    PubMed

    Martín, Javier; Odoom, Kofi; Tuite, Gráinne; Dunn, Glynis; Hopewell, Nicola; Cooper, Gill; Fitzharris, Catherine; Butler, Karina; Hall, William W; Minor, Philip D

    2004-12-01

    A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a "defective" molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.

  16. Long-Term Excretion of Vaccine-Derived Poliovirus by a Healthy Child

    PubMed Central

    Martín, Javier; Odoom, Kofi; Tuite, Gráinne; Dunn, Glynis; Hopewell, Nicola; Cooper, Gill; Fitzharris, Catherine; Butler, Karina; Hall, William W.; Minor, Philip D.

    2004-01-01

    A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a “defective” molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected. PMID:15564492

  17. [Investigation of a Patient with Pre-vaccine-derived Poliovirus in Shandong Province, China].

    PubMed

    Lin, Xiaojuan; Liu, Yao; Wang, Suting; Zhang Xiao; Song, Lizhi; Tao, Zexin; Ji, Feng; Xiong, Ping; Xu, Aiqiang

    2015-09-01

    To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.

  18. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

    NASA Technical Reports Server (NTRS)

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

  19. Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition

    PubMed Central

    Gustin, Kurt E.; Sarnow, Peter

    2001-01-01

    Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non-shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi-permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor–cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus-infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus. PMID:11226174

  20. Massive outbreak of poliomyelitis caused by type-3 wild poliovirus in Angola in 1999.

    PubMed Central

    Valente, F.; Otten, M.; Balbina, F.; Van de Weerdt, R.; Chezzi, C.; Eriki, P.; Van-Dúnnen, J.; Bele, J. M.

    2000-01-01

    The largest outbreak of poliomyelitis ever recorded in Africa (1093 cases) occurred from 1 March to 28 May 1999 in Luanda, Angola, and in surrounding areas. The outbreak was caused primarily by a type-3 wild poliovirus, although type-1 wild poliovirus was circulating in the outbreak area at the same time. Infected individuals ranged in age from 2 months to 22 years; 788 individuals (72%) were younger than 3 years. Of the 590 individuals whose vaccination status was known, 23% had received no vaccine and 54% had received fewer than three doses of oral poliovirus vaccine (OPV). The major factors that contributed to this outbreak were as follows: massive displacement of unvaccinated persons to urban settings; low routine OPV coverage; inaccessible populations during the previous three national immunization days (NIDs); and inadequate sanitation. This outbreak indicates the urgent need to improve accessibility to all children during NIDs and the dramatic impact that war can have by displacing persons and impeding access to routine immunizations. The period immediately after an outbreak provides an enhanced opportunity to eradicate poliomyelitis. If continuous access in all districts for acute flaccid paralysis surveillance and supplemental immunizations cannot be assured, the current war in Angola may threaten global poliomyelitis eradication. PMID:10812730

  1. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    SciTech Connect

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.

  2. Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: Evidence for phosphorylation

    SciTech Connect

    Ransone, L.J.; Dasgupta, A. )

    1989-11-01

    Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.

  3. Mechanisms of poliovirus inactivation by the direct and indirect effects of ionizing radiation

    SciTech Connect

    Ward, R.L.

    1980-08-01

    This study was designed to measure the effects of ionizing radiation on poliovirus particles when given under conditions where either direct (in broth) or indirect (in water) effects were predominant. Under direct conditions, inactivation of poliovirus was found to be due primarily to RNA damage, although capsid damage could account for about one-third of the viral inactivation. RNA damage did not appear to be due to strand breakage and therefore was probably caused primarily by base damage or crosslink formation. Capsid damage under direct irradiation conditions did not result in significant alterations of either the sedimentation coefficients or the isoelectric points of the poliovirus particles or detectable modification of the sizes of the viral proteins. It did, however, cause loss of availability to bind to host cells. Under indirect conditions no more than 25% of viral inactivation appeared to be due to RNA damage. However, the sedimentation coefficients and isoelectric points of the viral particles were greatly altered, and their abilities to bind to cells were lost at about three-fourths the rate of loss of infectivity. Capsid damage in this case did result in changes in the sizes of capsid proteins. Therefore, the majority of the radiation inactivation under indirect conditions appeared to be due to protein damage.

  4. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

    NASA Technical Reports Server (NTRS)

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

  5. Oral iodine supplementation does not reduce neutralizing antibody responses to oral poliovirus vaccine.

    PubMed Central

    Taffs, R. E.; Enterline, J. C.; Rusmil, K.; Muhilal; Suwardi, S. S.; Rustama, D.; Djatnika; Cobra, C.; Semba, R. D.; Cohen, N.; Asher, D. M.

    1999-01-01

    Iodine deficiency is a major cause of impaired mental development, goitre, and cretinism in many parts of the world. Because existing immunization programmes can be used to deliver oral iodized oil (OIO) to infants at risk, it was important to know whether OIO could adversely affect the antibody response to vaccines, such as trivalent oral poliovirus vaccine (OPV). A randomized, double-blind, placebo-controlled clinical trial was conducted in Subang, West Java, Indonesia, in which 617 eight-week-old infants received either OIO or a placebo (poppy-seed oil) during a routine visit for their first dose of OPV as part of the Expanded Programme on Immunization (EPI). The infants received two boosters of OPV at 4-week intervals after the first dose, and were followed up when 6 months old. Neutralizing antibody titres to poliovirus serotypes 1, 2, and 3 were compared in serum samples that were taken from 478 of these infants just before the first dose of OPV and at 6 months. It was found that oral iodized oil did not reduce the antibody responses to any of the three serotypes of OPV. These results indicate that oral iodine may safely be delivered to infants at the same time as oral poliovirus vaccine according to current EPI immunization schedules. PMID:10427933

  6. Mutational analysis of the genome-linked protein VPg of poliovirus.

    PubMed Central

    Kuhn, R J; Tada, H; Ypma-Wong, M F; Semler, B L; Wimmer, E

    1988-01-01

    Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal. Images PMID:2845132

  7. Expression of poliovirus 2Apro in mammalian cells: effects on translation.

    PubMed

    Aldabe, R; Feduchi, E; Novoa, I; Carrasco, L

    1995-12-11

    Poliovirus protease 2Apro has been efficiently expressed in HeLa and COS cells upon transfection with vector pTM1-2A and infection with the recombinant vaccinia virus bearing the T7 RNA polymerase. The expressed poliovirus protease localizes to the cytoplasm of the transfected cells, both in the endoplasmic reticulum and in vesicles scattered in the cytoplasm. Cleavage of p220, a component of initiation factor eIF-4F, selectively occurs from 5 h post-infection in transfected cells infected with the recombinant virus. This cleavage correlates in time with the profound inhibition observed in the synthesis of vaccinia virus proteins. A similar blockade of vesicular stomatitis virus translation takes place upon 2Apro expression. Finally, the synthesis of poliovirus protein 2C from a recombinant vaccinia virus that expresses this protein under the EMC untranslated leader region is not affected by the synthesis of 2Apro. These findings lend support to the idea that translation of capped mRNAs requires the integrity of p220, while this requirement is not observed when translation of a mRNA bearing a picornavirus leader region is assayed.

  8. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests.

    PubMed Central

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past. PMID:10743284

  9. Mapping of the epitopes of poliovirus type 2 in complex with antibodies.

    PubMed

    Bannwarth, Ludovic; Girerd-Chambaz, Yves; Arteni, Ana; Guigner, Jean-Michel; Ronzon, Frederic; Manin, Catherine; Vénien-Bryan, Catherine

    2015-10-01

    The inactivated polio vaccine (IPV) contains poliovirus (PV) samples that belong to serotypes 1, 2 and 3. All three serotypes contain the D-antigen, which induces protective antibodies. The antigenic structure of PVs consists of at least four different antigenic sites and the D-antigen content represents the combined activity of multiple epitopes (Ferguson et al., 1993; Minor, 1990; Minor et al., 1986). The potency of IPV vaccines is determined by measuring the D-antigen content. Several ELISA methods have been developed using polyclonal or monoclonal antibodies (Mabs) in order to quantify the D-antigen content. Characterization of the epitopes recognized by the different Mabs is crucial to map the entire virus surface and ensure the presence of epitopes able to induce neutralizing antibodies. Using a new approach that we developed to study the interaction between monoclonal antibodies and poliovirus type 2, which combines cryo-electron microscopy, image analysis and X-ray crystallography along with identification of exposed amino acids, we have mapped in 3D the epitope sites recognized by three specific Fabs at the surface of poliovirus type 2 (PV2) and characterized precisely the antigenic sites for these Fabs.

  10. National laboratory inventories for wild poliovirus containment--Western Pacific region, 2008.

    PubMed

    2009-09-11

    In the future, when wild poliovirus (WPV) transmission is interrupted worldwide, facilities holding WPV materials will represent the only remaining repository of the virus. Maintaining the number of such facilities at a minimum and at an appropriate biosafety standard (laboratory containment) reduces the risk for a facility-associated reintroduction of WPV. In May 1999, the World Health Assembly (WHA) urged all member states to begin the process leading to laboratory containment of WPV. The World Health Organization (WHO) global action plan for laboratory containment of WPV issued in 1999 indicated a staged approach that begins with a national survey of all biomedical facilities (Phase I); the purpose of the survey is to alert institutions and facilities to the need for containment, encourage reduction of WPV materials, and develop a national inventory of facilities holding such materials. The survey and inventory provide a facility database for use in all subsequent steps toward global poliovirus containment. In May 2008, WHA urged all WHO member states to complete Phase I activities outlined in the WHO Global Action Plan for Laboratory Containment of Wild Polioviruses. In the WHO Western Pacific Region (WPR), Phase I surveys of 77,260 laboratories in the 37 countries and areas of WPR were conducted during 1999--2008. A total of 45 laboratories were identified as holding WPV materials in 2008. This report describes completion of Phase I containment activities by WPR countries, and updates a previous report on Phase I completion in the European Region and global progress.

  11. Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges.

    PubMed Central

    Pancorbo, O C; Bitton, G; Farrah, S R; Gifford, G E; Overman, A R

    1988-01-01

    The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns. PMID:2830848

  12. Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

    PubMed

    La Monica, N; Meriam, C; Racaniello, V R

    1986-02-01

    Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.

  13. Molecular epidemiology of silent introduction and sustained transmission of wild poliovirus type 1, Israel, 2013.

    PubMed

    Shulman, L M; Gavrilin, E; Jorba, J; Martin, J; Burns, C C; Manor, Y; Moran-Gilad, J; Sofer, D; Hindiyeh, M Y; Gamzu, R; Mendelson, E; Grotto, I

    2014-02-20

    Poliovirus vaccine coverage in Israel is over 90%. The last nine birth cohorts have been vaccinated exclusively with inactivated polio vaccine (IPV). However, between February and July 2013 type 1 wild poliovirus (WPV1) was detected persistently in 10 and intermittently in 8 of 47 environmental surveillance sites in southern and central Israel and in 30 stool samples collected during July from healthy individuals in southern Israel. We report results of sequence and phylogenetic analyses of genes encoding capsid proteins to determine the source and transmission mode of the virus. WPV1 capsid protein 1 nucleotide sequences were most closely related to South Asia (SOAS) cluster R3A polioviruses circulating in Pakistan in 2012 and isolated from Egyptian sewage in December 2012. There was no noticeable geographical clustering within WPV1-positive sites. Uniform codon usage among isolates from Pakistan, Egypt and Israel showed no signs of optimisation or deoptimisation. Bayesian phylogenetic time clock analysis of the entire capsid coding region (2,643 nt) with a 1.1% evolutionary rate indicated that Israeli and Egyptian WPV1-SOAS lineages diverged in September 2012, while Israeli isolates split into two sub-branches after January 2013. This suggests one or more introduction events into Israel with subsequent silent circulation despite high population immunity.

  14. Some characteristics of poliovirus strains isolated in Uganda between 1966 and 1971.

    PubMed

    Balayan, M S; Dömök, I; Fayinka, O A; Soneji, A D

    1976-01-01

    Sixty-five poliovirus strains were investigated in genetic marker tests in order to obtain information on the characteristics of polioviruses circulating in Uganda where, owing to the insufficient use of live poliovirus vaccine, poliomyelitis remained a serious public health problem. Of the type 1 strains predominant in both epidemic and non-epidemic years, 29 were studied for their antigenic fine structure. Based on their intratypic character, these strains proved to represent six different antigenic variants. Three of these variants were predominant during certain periods; the first variant was present in 1966 and 1968, the second in 1967, and the third from 1969 to the end of observation period. Four strains from Kuwait and three from Ghana isolated in 1969 and 1970 showed an antigenic structure identical to that of the strains predominant in Uganda in these years. Some strains proved to be of vaccine origin. Twenty-nine type 1 and 24 type 2 strains showed a great variety of characteristics when studied in d, od, and rct/40 marker tests. There was no indication that the distribution of strains according to their in vitro markers would have been different in epidemic and non-epidemic years, or that any particular combination of markers would have been more common among strains isolated from paralytic patients than among those from non-paralytic patients. Nine of 12 type 3 strains had the rct/40(+) marker.

  15. Some characteristics of poliovirus strains isolated in Uganda between 1966 and 1971*

    PubMed Central

    Balayan, M. S.; Dömök, I.; Fayinka, O. A.; Soneji, A. D.

    1976-01-01

    Sixty-five poliovirus strains were investigated in genetic marker tests in order to obtain information on the characteristics of polioviruses circulating in Uganda where, owing to the insufficient use of live poliovirus vaccine, poliomyelitis remained a serious public health problem. Of the type 1 strains predominant in both epidemic and non-epidemic years, 29 were studied for their antigenic fine structure. Based on their intratypic character, these strains proved to represent six different antigenic variants. Three of these variants were predominant during certain periods; the first variant was present in 1966 and 1968, the second in 1967, and the third from 1969 to the end of observation period. Four strains from Kuwait and three from Ghana isolated in 1969 and 1970 showed an antigenic structure identical to that of the strains predominant in Uganda in these years. Some strains proved to be of vaccine origin. Twenty-nine type 1 and 24 type 2 strains showed a great variety of characteristics when studied in d, od, and rct/40 marker tests. There was no indication that the distribution of strains according to their in vitro markers would have been different in epidemic and non-epidemic years, or that any particular combination of markers would have been more common among strains isolated from paralytic patients than among those from non-paralytic patients. Nine of 12 type 3 strains had the rct/40+ marker. PMID:186208

  16. An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector

    PubMed Central

    Perri, Silvia; Greer, Catherine E.; Thudium, Kent; Doe, Barbara; Legg, Harold; Liu, Hong; Romero, Raul E.; Tang, Zequn; Bin, Qian; Dubensky, Thomas W.; Vajdy, Michael; Otten, Gillis R.; Polo, John M.

    2003-01-01

    Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety. PMID:12970424

  17. In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs ▿ ‡

    PubMed Central

    Kamrud, Kurt I.; Coffield, V. McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A.; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K.; Berglund, Peter; Smith, Jonathan

    2010-01-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans. PMID:20504925

  18. Effects of activated aflatoxin B/sub 1/ and caffeine on DNA replicon initiation in HeLa cells

    SciTech Connect

    Cramer, P.; Painter, R.B.

    1981-01-01

    Afatoxin B/sub 1/ (AFB/sub 1/) is activated by a rat microsomal extract (S-9) to form a product that inhibits DNA synthesis in HeLa cells. At 10/sup -7/ M, AFB/sub 1/ inhibited initiation of replicons, as shown in alkaline sucrose gradient profiles 30 min after incubation with the drug. Ninety minutes later, the profile of treated cells was similar to that of control, but 4 h later there was another effect on replicon initiation. At 10/sup -6/ M, the inhibition of initiation was greater than at 10/sup -7/ M and increased progressively. Four hours after removal of the drug, the gradient profile showed low amounts of radioactivity in all size classes of DNA. When cells were incubated in medium containing caffeine (2 mM) even as late as 60 min after incubation with AFB/sub 1/, the inhibition of replicon initiation was prevented. If caffeine was later removed from the medium, replicon initiation was then inhibited. At 10/sup -7/ M or 10/sup -6/ M, AFB/sub 1/ had little immediate effect on chain elongation, but at 10/sup -5/ M, the gradient profiles showed an accumulation of low molecular weight DNA molecules, with no radioactivity in the region of high molecular weight DNA, owing to a block to chain elongation; this was not affected by caffeine. These results suggest that AFB/sub 1/ induces damage that changes the fonformation of chromatin so that initiation of new replicons cannot occur; in the presence of caffeine this change does not occur and DNA replication is not inhibited.

  19. Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses.

    PubMed

    Pushko, P; Geisbert, J; Parker, M; Jahrling, P; Smith, J

    2001-12-01

    Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.

  20. A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

    PubMed Central

    Barco, Angel; Feduchi, Elena; Carrasco, Luis

    2000-01-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

  1. Environmental surveillance of poliovirus and non-polio enterovirus in urban sewage in Dakar, Senegal (2007-2013)

    PubMed Central

    Ndiaye, Abdou Kader; Diop, Pape Amadou Mbathio; Diop, Ousmane Madiagne

    2014-01-01

    Introduction Global poliomyelitis eradication initiative relies on (i) laboratory based surveillance of acute flaccid surveillance (AFP) to monitor the circulation of wild poliovirus in a population, and (ii) vaccination to prevent its diffusion. However, as poliovirus can survive in the environment namely in sewage, environmental surveillance (ES) is of growing importance as the eradication target is close. This study aimed to assess polioviruses and non polio enteroviruses circulation in sewage drains covering a significant population of Dakar. Methods From April 2007 to May 2013, 271 specimens of raw sewage were collected using the grab method in 6 neighborhoods of Dakar. Samples were processed to extract and concentrate viruses using polyethylene glycol and Dextran (two-phase separation method). Isolation of enteroviruses was attempted in RD, L20B and Hep2 cell lines. Polioviruses were identified by RT-PCR and Elisa. Non Polio Enteroviruses (NPEVs) were identified by RT-PCR and microneutralisation tests. Results Polioviruses and NPEVs were respectively detected in 34,3% and 42,8% sewage samples. No wild poliovirus neither circulating vaccine-derived Poliovirus (cVDPV) was detected. Neutralization assays have identified 49 non polio enteroviruses that were subsequently classified in 13 serotypes belonging to HEV-A (22, 4%), HEV-B (12, 24%), HEV-C (26, 53%) and HEV-D (6, 12%) species. Conclusion This study is the first documentation of enteroviruses environmental detection in Senegal. It shows the usefulness of environmental surveillance for indirect monitoring of the circulation and distribution of enteroviruses in the community. PMID:25848458

  2. [Genetic recombination in vaccine poliovirus: comparative study in strains excreted in course of vaccination by oral poliovirus vaccine and circulating strains].

    PubMed

    Haddad-Boubaker, S; Ould-Mohamed-Abdallah, M V; Ben-Yahia, A; Triki, H

    2010-12-01

    Recombination is one of the major mechanisms of evolution in poliovirus. In this work, recombination was assessed in children during vaccination with OPV and among circulating vaccine strains isolated in Tunisia during the last 15 years in order to identify a possible role of recombination in the response to the vaccine or the acquisition of an increased transmissibility. This study included 250 poliovirus isolates: 137 vaccine isolates, excreted by children during primary vaccination with OPV and 113 isolates obtained from acute flaccid paralytic (AFP) cases and healthy contacts. Recombination was first assessed using a double PCR-RFLP, and sequencing. Nineteen per cent of recombinant strains were identified: 20% of strains excreted by vaccinees among 18% of circulating strains. The proportion of recombinant in isolates of serotype1 was very low in the two groups while the proportions of recombinants in serotypes 2 and 3 were different. In vaccinees, the frequency of recombinants in serotype3 decreased during the course of vaccination: 54% after the first dose, 32% after the second and 14% after the third dose. These results suggest that recombination enhances the ability of serotype3 vaccine strains to induce an immune response. Apart from recent vaccination, it may contribute to a more effective transmissibility of vaccine strains among human population. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  3. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

    Phua, Kyle K L; Leong, Kam W; Nair, Smita K

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  4. Naked mole rats exhibit metabolic but not ventilatory plasticity following chronic sustained hypoxia.

    PubMed

    Chung, Danielle; Dzal, Yvonne A; Seow, Allison; Milsom, William K; Pamenter, Matthew E

    2016-03-30

    Naked mole rats are among the most hypoxia-tolerant mammals identified and live in chronic hypoxia throughout their lives. The physiological mechanisms underlying this tolerance, however, are poorly understood. Most vertebrates hyperventilate in acute hypoxia and exhibit an enhanced hyperventilation following acclimatization to chronic sustained hypoxia (CSH). Conversely, naked mole rats do not hyperventilate in acute hypoxia and their response to CSH has not been examined. In this study, we explored mechanisms of plasticity in the control of the hypoxic ventilatory response (HVR) and hypoxic metabolic response (HMR) of freely behaving naked mole rats following 8-10 days of chronic sustained normoxia (CSN) or CSH. Specifically, we investigated the role of the major inhibitory neurotransmitter γ-amino butyric acid (GABA) in mediating these responses. Our study yielded three important findings. First, naked mole rats did not exhibit ventilatory plasticity following CSH, which is unique among adult animals studied to date. Second, GABA receptor (GABAR) antagonism altered breathing patterns in CSN and CSH animals and modulated the acute HVR in CSN animals. Third, naked mole rats exhibited GABAR-dependent metabolic plasticity following long-term hypoxia, such that the basal metabolic rate was approximately 25% higher in normoxic CSH animals than CSN animals, and GABAR antagonists modulated this increase.

  5. Naked mole rats exhibit metabolic but not ventilatory plasticity following chronic sustained hypoxia

    PubMed Central

    Chung, Danielle; Dzal, Yvonne A.; Seow, Allison; Milsom, William K.; Pamenter, Matthew E.

    2016-01-01

    Naked mole rats are among the most hypoxia-tolerant mammals identified and live in chronic hypoxia throughout their lives. The physiological mechanisms underlying this tolerance, however, are poorly understood. Most vertebrates hyperventilate in acute hypoxia and exhibit an enhanced hyperventilation following acclimatization to chronic sustained hypoxia (CSH). Conversely, naked mole rats do not hyperventilate in acute hypoxia and their response to CSH has not been examined. In this study, we explored mechanisms of plasticity in the control of the hypoxic ventilatory response (HVR) and hypoxic metabolic response (HMR) of freely behaving naked mole rats following 8–10 days of chronic sustained normoxia (CSN) or CSH. Specifically, we investigated the role of the major inhibitory neurotransmitter γ-amino butyric acid (GABA) in mediating these responses. Our study yielded three important findings. First, naked mole rats did not exhibit ventilatory plasticity following CSH, which is unique among adult animals studied to date. Second, GABA receptor (GABAR) antagonism altered breathing patterns in CSN and CSH animals and modulated the acute HVR in CSN animals. Third, naked mole rats exhibited GABAR-dependent metabolic plasticity following long-term hypoxia, such that the basal metabolic rate was approximately 25% higher in normoxic CSH animals than CSN animals, and GABAR antagonists modulated this increase. PMID:27009224

  6. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  7. Development and application of West Nile virus subgenomic replicon RNA expressing secreted alkaline phosphatase.

    PubMed

    Moritoh, Kanako; Maeda, Akihiko; Nishino, Tomohiro; Sasaki, Nobuya; Agui, Takashi

    2011-05-01

    We have developed a West Nile virus (WNV) subgenomic replicon harboring the secreted alkaline phosphatase (SEAP) reporter gene instead of viral structural genes (designated repWNV/SEAP). The repWNV/SEAP allowed easy evaluation of viral replication efficiency by direct measurement of SEAP secretion in the cell culture medium in physical containment level 2 facilities. Furthermore, we validated the availability of this system using a known anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). The Oas1b-transfected cells were more resistant to repWNV/SEAP replication than the original cells. Thus, this system not only affords a useful tool for identification/evaluation of anti-flavivirus genes/drugs in terms of safety, ease of use and reliability, but should be able to reduce or replace the bioassay using laboratory animals.

  8. Predictive spatial risk model of poliovirus to aid prioritization and hasten eradication in Nigeria

    PubMed Central

    2014-01-01

    Background One of the challenges facing the Global Polio Eradication Initiative is efficiently directing limited resources, such as specially trained personnel, community outreach activities, and satellite vaccinator tracking, to the most at-risk areas to maximize the impact of interventions. A validated predictive model of wild poliovirus circulation would greatly inform prioritization efforts by accurately forecasting areas at greatest risk, thus enabling the greatest effect of program interventions. Methods Using Nigerian acute flaccid paralysis surveillance data from 2004-2013, we developed a spatial hierarchical Poisson hurdle model fitted within a Bayesian framework to study historical polio caseload patterns and forecast future circulation of type 1 and 3 wild poliovirus within districts in Nigeria. A Bayesian temporal smoothing model was applied to address data sparsity underlying estimates of covariates at the district level. Results We find that calculated vaccine-derived population immunity is significantly negatively associated with the probability and number of wild poliovirus case(s) within a district. Recent case information is significantly positively associated with probability of a case, but not the number of cases. We used lagged indicators and coefficients from the fitted models to forecast reported cases in the subsequent six-month periods. Over the past three years, the average predictive ability is 86 ± 2% and 85 ± 4% for wild poliovirus type 1 and 3, respectively. Interestingly, the predictive accuracy of historical transmission patterns alone is equivalent (86 ± 2% and 84 ± 4% for type 1 and 3, respectively). We calculate uncertainty in risk ranking to inform assessments of changes in rank between time periods. Conclusions The model developed in this study successfully predicts districts at risk for future wild poliovirus cases in Nigeria. The highest predicted district risk was 12.8 WPV1 cases in 2006, while the lowest district risk

  9. Prime-boost vaccinations using recombinant flavivirus replicon and vaccinia virus vaccines: an ELISPOT analysis.

    PubMed

    Rattanasena, Paweena; Anraku, Itaru; Gardner, Joy; Le, Thuy T; Wang, Xiang Ju; Khromykh, Alexander A; Suhrbier, Andreas

    2011-03-01

    Recombinant Kunjin replicon virus-like particle (VLP), vaccinia virus (rVV) and DNA vaccines were tested in a large series of prime-boost vaccinations using interferon (IFN)γ ELISPOT assays that reflected effector (E), effector memory (EM) and central memory (CM) responses. All vaccine constructs encoded the murine polytope immunogen and responses to four CD8 T-cell epitopes (TYQRTRALV, SYIPSAEKI, YPHFMPTNL and RPQASGVYM) were measured. VLP/rVV out performed (by 14- to 20-fold) DNA/rVV for induction of CM responses, whereas EM responses were only marginally increased. DNA/VLP induced more EM, but not CM responses, than VLP alone, illustrating that DNA priming is not universally beneficial. rVV/VLP gave comparable results to VLP/rVV combinations, although the former induced approximately threefold more E responses, illustrating the utility of poxvirus priming in this setting. Although higher doses of VLP and rVV increased responses after single immunizations, such dose increases provided only marginal benefit in heterologous prime-boost settings. Triple combinations also provided no benefit over two vaccinations. DNA vaccination was associated with broad CM, but not EM responses, and the breadth of EM and E responses was significantly improved by increasing viral vector dose. VLP/rVV, rather than DNA priming, induced T cells with consistently high IFNγ secretion profiles across all ELISPOT measures. Vector-specific CD8 T-cell responses generally correlated well with immunogen-specific responses, although, as expected, single use of each vector reduced the relative levels of vector-specific responses. These experiments illustrate the utility of replicons in heterologous prime-boost vaccinations, and illustrate the diversity of data that can be obtained from ELISPOT analyses.

  10. The naked T Tauri stars - The low-mass pre-main sequence unveiled

    NASA Technical Reports Server (NTRS)

    Walter, Frederick M.

    1987-01-01

    The search for low-mass premain-sequence (PMS) stars associated with X-ray sources in regions of star formation is discussed. The survey to date has revealed at least 30 low-mass PMS stars in the Tau-Aur region, and a comparable number in Oph. These stars are the naked T Tau stars, unveiled versions of the well-known classical T Tau stars. The properties of these newly discovered PMS stars and their relation to the classical T Tau stars are discussed, and it is concluded that the naked T Tau stars are the true low-mass PMS stars, and that the observable characteristics defining the classical T Tau stars are due to the interaction of an underlying, fairly normal star with a dominant circumstellar environment. The impact the naked T Tau stars are likely to have on models of the PMS evolution of low-mass stars is considered.

  11. The MTSU Uranidrome: A Naked-Eye Observatory for Teaching Astronomy and Geometry

    NASA Astrophysics Data System (ADS)

    Klumpe, Eric W.

    2006-12-01

    Prior to the invention of the telescope astronomy was done with the naked eye. The astronomical models that ancient astronomers were able to construct based upon their naked-eye observations are impressive. Following those examples, Middle Tennessee State University has built a 30-meter diameter, naked-eye, observatory to serve as a hands-on teaching tool for astronomy and geometry. The primary goal is for astronomy students to use their own measurements to develop accurate models of how our solar system works and to better appreciate the creativity of ancient astronomers. Typical Uranidrome activities include measuring the axial tilt of the Earth, the latitude and longitude of MTSU, the spin rate and orbital rate of the Earth, the circumference of the Earth, true-north relative to magnetic-north, short-term predictions of eclipses, and using the Sun to determine the date and time. The Uranidrome was funded by grants from NASA and Middle Tennessee State University.

  12. The naked T Tauri stars - The low-mass pre-main sequence unveiled

    NASA Technical Reports Server (NTRS)

    Walter, Frederick M.

    1987-01-01

    The search for low-mass premain-sequence (PMS) stars associated with X-ray sources in regions of star formation is discussed. The survey to date has revealed at least 30 low-mass PMS stars in the Tau-Aur region, and a comparable number in Oph. These stars are the naked T Tau stars, unveiled versions of the well-known classical T Tau stars. The properties of these newly discovered PMS stars and their relation to the classical T Tau stars are discussed, and it is concluded that the naked T Tau stars are the true low-mass PMS stars, and that the observable characteristics defining the classical T Tau stars are due to the interaction of an underlying, fairly normal star with a dominant circumstellar environment. The impact the naked T Tau stars are likely to have on models of the PMS evolution of low-mass stars is considered.

  13. Accessing the Genomic Effects of Naked Nanoceria in Murine Neuronal Cells

    PubMed Central

    Lee, Tin-Lap; Raitano, Joan M.; Rennert, Owen M; Chan, Siu-Wai; Chan, Wai-Yee

    2011-01-01

    Cerium oxide nanoparticles (nanoceria) are versatile engineered nanoparticles (ENPs) due to their unique redox properties. We and others have previously demonstrated naked nanoceria could act as antioxidants to protect cells against oxidative damage. While the redox properties may be beneficial, the genome-wide effects of nanoceria on gene transcription and associated biological processes remain elusive. Here we applied functional genomic approach to examine the genome-wide effects of nanoceria on global gene transcription and cellular functions in mouse neuronal cells. Importantly, we demonstrated nanoceria induced chemical- and size-specific changes in the murine neuronal cell transcriptome. The nanoceria specially contributed more than 83% of uniquely altered gene population and associate with a unique spectrum of genes related to neurological disease, cell cycle control and growth. These observations suggest an in-depth assessment of potential health effects of naked nanoceria and other naked nanoparticles is both necessary and imminent. PMID:21889474

  14. The value of naked eye examination of biopsied lymph nodes in the diagnosis of tuberculous lymphadenitis.

    PubMed

    Bem, C

    1996-01-01

    Tuberculous lymphadenitis is common in Central Africa, where diagnosis by histological examination of a biopsied node is often delayed. In the present study, the naked eye appearance of the cut surface of 306 consecutive biopsied lymph nodes was compared with the histological diagnosis. One hundred and eight-eight nodes showed tuberculosis on histology (including two with coexisting second pathology). One hundred and forty-eight (79%) cases of tuberculous lymphadenitis (including both with coexisting second pathology) showed noncaseating tuberculomata or caseation visible on naked eye examination. Such signs were not seen in other nodes. Other signs were seen in another 18 (10%) tuberculous nodes. It is concluded that naked eye examination of nodes provides useful information for the diagnosis of tuberculous lymphadenitis, pending confirmation by histology.

  15. Attenuation of neurovirulence, biodistribution, and shedding of a poliovirus:rhinovirus chimera after intrathalamic inoculation in Macaca fascicularis.

    PubMed

    Dobrikova, Elena Y; Goetz, Christian; Walters, Robert W; Lawson, Sarah K; Peggins, James O; Muszynski, Karen; Ruppel, Sheryl; Poole, Karyol; Giardina, Steven L; Vela, Eric M; Estep, James E; Gromeier, Matthias

    2012-03-01

    A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.

  16. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.

    PubMed Central

    Pelletier, J; Kaplan, G; Racaniello, V R; Sonenberg, N

    1988-01-01

    Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation. Images PMID:2835660

  17. Outbreak of Type 2 Vaccine-Derived Poliovirus in Nigeria: Emergence and Widespread Circulation in an Underimmunized Population

    PubMed Central

    Pate, Muhammad Ali; Wannemuehler, Kathleen; Jenks, Julie; Burns, Cara; Chenoweth, Paul; Abanida, Emmanuel Ade; Adu, Festus; Baba, Marycelin; Gasasira, Alex; Iber, Jane; Mkanda, Pascal; Williams, A. J.; Shaw, Jing; Pallansch, Mark; Kew, Olen

    2011-01-01

    Wild poliovirus has remained endemic in northern Nigeria because of low coverage achieved in the routine immunization program and in supplementary immunization activities (SIAs). An outbreak of infection involving 315 cases of type 2 circulating vaccine-derived poliovirus (cVDPV2; >1% divergent from Sabin 2) occurred during July 2005–June 2010, a period when 23 of 34 SIAs used monovalent or bivalent oral poliovirus vaccine (OPV) lacking Sabin 2. In addition, 21 “pre-VDPV2” (0.5%–1.0% divergent) cases occurred during this period. Both cVDPV and pre-VDPV cases were clinically indistinguishable from cases due to wild poliovirus. The monthly incidence of cases increased sharply in early 2009, as more children aged without trivalent OPV SIAs. Cumulative state incidence of pre-VDPV2/cVDPV2 was correlated with low childhood immunization against poliovirus type 2 assessed by various means. Strengthened routine immunization programs in countries with suboptimal coverage and balanced use of OPV formulations in SIAs are necessary to minimize risks of VDPV emergence and circulation. PMID:21402542

  18. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region

    SciTech Connect

    Pelletier, J.; Kaplan, G.; Racaniello, V.R.; Sonenberg, N.

    1988-03-01

    Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, the authors determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. They found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. They also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.

  19. Stool screening of Syrian refugees and asylum seekers in Germany, 2013/2014: Identification of Sabin like polioviruses.

    PubMed

    Böttcher, Sindy; Neubauer, Katrin; Baillot, Armin; Rieder, Gabriele; Adam, Maja; Diedrich, Sabine

    2015-10-01

    Germany is a partner of the Global Polio Eradication Initiative. Assurance of polio free status is based on enterovirus surveillance, which focuses on patients with signs of acute flaccid paralysis or aseptic meningitis/encephalitis, representing the key symptoms of poliovirus infection. In response to the wild poliovirus outbreak in Syria 2013 and high number of refugees coming from Syria to Germany, stool samples from 629 Syrian refugees/asylum seekers aged <3 years were screened for wild poliovirus between November 2013 and April 2014. Ninety-three samples (14.8%) were positive in an enterovirus specific PCR. Of these, 12 contained Sabin-like polioviruses. The remaining 81 samples were characterized as non-polio enteroviruses representing several members of groups A-C as well as rhinovirus. Wild-type poliovirus was not detected via stool screening involving molecular and virological methods, indicating a very low risk for the importation by Syrian refugees and asylum seekers at that time. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. Circulating type 1 vaccine-derived poliovirus may evolve under the pressure of adenosine deaminases acting on RNA.

    PubMed

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Xu, Ruixue; Wang, Shujing

    2015-01-01

    Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution.

  1. Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

    PubMed Central

    Dobrikova, Elena Y.; Goetz, Christian; Walters, Robert W.; Lawson, Sarah K.; Peggins, James O.; Muszynski, Karen; Ruppel, Sheryl; Poole, Karyol; Giardina, Steven L.; Vela, Eric M.; Estep, James E.

    2012-01-01

    A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied. PMID:22171271

  2. Outbreak of paralytic poliomyelitis in Finland: widespread circulation of antigenically altered poliovirus type 3 in a vaccinated population.

    PubMed

    Hovi, T; Cantell, K; Huovilainen, A; Kinnunen, E; Kuronen, T; Lapinleimu, K; Pöyry, T; Roivainen, M; Salama, N; Stenvik, M

    1986-06-21

    An outbreak of 9 cases of paralytic poliomyelitis and 1 non-paralytic case occurred in Finland between August, 1984, and January, 1985, after two decades of freedom from the disease attributable to a successful immunisation programme. During the outbreak poliovirus type 3 was isolated from the patients, from about 15% of healthy persons tested, and from sewage water. At least 100 000 persons were estimated to have been infected. With 1.5 million extra doses of inactivated poliovirus vaccine to children under 18 years of age and an oral poliovirus vaccine campaign covering about 95% of the entire population in February-March, 1985, the outbreak was halted in February, 1985. Impaired herd immunity to the epidemic strain of poliovirus type 3, which differed from the type 3 vaccine strains in both immunological and molecular properties, was important in the emergence of this outbreak. The inactivated poliovaccine that had been used in the vaccination programme was relatively weakly immunogenic, especially as regards the type 3 component. Whether continuous antigenic variation of poliovirus type 3 has wider epidemiological implications is not known.

  3. Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.

    PubMed

    Tamura, Masato; Kawano, Mitsuoki; Sato, Mari; Nashimoto, Masayuki

    2015-10-01

    tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl‑β‑cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis‑inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin‑, caveolae‑ and raft‑independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis‑inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA‑mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically‑targeted and potent sgRNA drugs.

  4. The Naked Mole-Rat Response to Oxidative Stress: Just Deal with It

    PubMed Central

    Lewis, Kaitlyn N.; Andziak, Blazej; Yang, Ting

    2013-01-01

    Abstract Significance: The oxidative stress theory of aging has been the most widely accepted theory of aging providing insights into why we age and die for over 50 years, despite mounting evidence from a multitude of species indicating that there is no direct relationship between reactive oxygen species (ROS) and longevity. Here we explore how different species, including the longest lived rodent, the naked mole-rat, have defied the most predominant aging theory. Recent Advances: In the case of extremely long-lived naked mole-rat, levels of ROS production are found to be similar to mice, antioxidant defenses unexceptional, and even under constitutive conditions, naked mole-rats combine a pro-oxidant intracellular milieu with high, steady state levels of oxidative damage. Clearly, naked mole-rats can tolerate this level of oxidative stress and must have mechanisms in place to prevent its translation into potentially lethal diseases. Critical Issues: In addition to the naked mole-rat, other species from across the phylogenetic spectrum and even certain mouse strains do not support this theory. Moreover, overexpressing or knocking down antioxidant levels alters levels of oxidative damage and even cancer incidence, but does not modulate lifespan. Future Directions: Perhaps, it is not oxidative stress that modulates healthspan and longevity, but other cytoprotective mechanisms that allow animals to deal with high levels of oxidative damage and stress, and nevertheless live long, relatively healthy lifespans. Studying these mechanisms in uniquely long-lived species, like the naked mole-rat, may help us tease out the key contributors to aging and longevity. Antioxid. Redox Signal. 19, 1388–1399. PMID:23025341

  5. Poliovirus 5′-Terminal Cloverleaf RNA Is Required in cis for VPg Uridylylation and the Initiation of Negative-Strand RNA Synthesis

    PubMed Central

    Lyons, Traci; Murray, Kenneth E.; Roberts, Allan W.; Barton, David J.

    2001-01-01

    Chimeric poliovirus RNAs, possessing the 5′ nontranslated region (NTR) of hepatitis C virus in place of the 5′ NTR of poliovirus, were used to examine the role of the poliovirus 5′ NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3′ NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5′-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5′-terminal cloverleaf and 3′ NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA. PMID:11602711

  6. Dynamics of a charged particle around a weakly magnetized naked singularity

    NASA Astrophysics Data System (ADS)

    Babar, Gulmina Zaman; Jamil, Mubasher; Lim, Yen-Kheng

    2016-12-01

    We examine the motion of a charged particle in the vicinity of a weakly magnetized naked singularity. The escape velocity and energy of the particle moving around the naked singularity after being kicked by another particle or photon are investigated. Also at innermost stable circular orbit (ISCO) escape velocity and energy are examined. Effective potential and angular momentum of the particle are also discussed. We discuss the center-of-mass energy after collision between two particles having same mass and opposite charges moving along the same circular orbit in the opposite direction. It is investigated that under what conditions maximum energy can be produced as a result of collision.

  7. Penetration into Host Cells of Naked, Partially Injected (Post-FST) DNA of Bacteriophage T5

    PubMed Central

    Labedan, B.; Legault-Demare, J.

    1973-01-01

    The experiments presented here suggest that the naked post-FST DNA, attached to the bacteria after a centrifugation at 6,000 × g of Escherichia coli-T5 complexes arrested at the FST stage, can be injected into the host cell if protein synthesis is allowed. This results from the observation that the production of infectious centers by this naked DNA is sensitive to DNase or moderate shearing treatments. The process of injection seems to be the same as that for DNA normally rolled in an adsorbed capsid. PMID:4583885

  8. Sex-specific but not sexually explicit: pupillary responses to dressed and naked adults

    PubMed Central

    Bindemann, Markus

    2017-01-01

    Dilation of the pupils is an indicator of an observer's sexual interest in other people, but it remains unresolved whether this response is strengthened or diminished by sexually explicit material. To address this question, this study compared pupillary responses of heterosexual men and women to naked and dressed portraits of male and female adult film actors. Pupillary responses corresponded with observers' self-reported sexual orientation, such that dilation occurred during the viewing of opposite-sex people, but were comparable for naked and dressed targets. These findings indicate that pupillary responses provide a sex-specific measure, but are not sensitive to sexually explicit content. PMID:28572991

  9. Sporadic Isolation of Sabin-Like Polioviruses and High-Level Detection of Non-Polio Enteroviruses during Sewage Surveillance in Seven Italian Cities, after Several Years of Inactivated Poliovirus Vaccination

    PubMed Central

    Battistone, A.; Buttinelli, G.; Fiore, S.; Amato, C.; Bonomo, P.; Patti, A. M.; Vulcano, A.; Barbi, M.; Binda, S.; Pellegrinelli, L.; Tanzi, M. L.; Affanni, P.; Castiglia, P.; Germinario, C.; Mercurio, P.; Cicala, A.; Triassi, M.; Pennino, F.

    2014-01-01

    Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5′ noncoding region (5′NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile > Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing. PMID:24814793

  10. Sporadic isolation of sabin-like polioviruses and high-level detection of non-polio enteroviruses during sewage surveillance in seven Italian cities, after several years of inactivated poliovirus vaccination.

    PubMed

    Battistone, A; Buttinelli, G; Fiore, S; Amato, C; Bonomo, P; Patti, A M; Vulcano, A; Barbi, M; Binda, S; Pellegrinelli, L; Tanzi, M L; Affanni, P; Castiglia, P; Germinario, C; Mercurio, P; Cicala, A; Triassi, M; Pennino, F; Fiore, L

    2014-08-01

    Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5'noncoding region (5'NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile>Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing.

  11. Multiagent Vaccines Vectored by Venezuelan Equine Encephalitis Virus Replicon Elicits Immune Responses to Marburg Virus and Protection Against Anthrax and Botulinum Neurotoxin in Mice

    DTIC Science & Technology

    2006-01-01

    formulations of individual Venezuelan equine encephalitis (VEE) virus replicon- vectored vaccines against a bacterial disease, anthrax; a viral disease...here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease...on days 0, 35, and 70 with the indicated vaccines. Ne b Infectious units were used to measure VRP and milliliters were used to measur c The

  12. Silent reintroduction of wild-type poliovirus to Israel, 2013 - risk communication challenges in an argumentative atmosphere.

    PubMed

    Kaliner, E; Moran-Gilad, J; Grotto, I; Somekh, E; Kopel, E; Gdalevich, M; Shimron, E; Amikam, Y; Leventhal, A; Lev, B; Gamzu, R

    2014-02-20

    Israel has been certified as polio-free by the World Health Organization and its routine immunisation schedule consists of inactivated poliovirus vaccine (IPV) only. At the end of May 2013, the Israeli Ministry of Health (MOH) has confirmed the reintroduction of wild-type poliovirus 1 into the country. Documented ongoing human-to-human transmission necessitated a thorough risk assessment followed by a supplemental immunisation campaign using oral polio vaccine (OPV). The unusual situation in which ongoing poliovirus transmission was picked up through an early warning system of sewage monitoring without active polio cases, brought about significant challenges in risk communication. This paper reviews the challenges faced by the MOH and the communication strategy devised, in order to facilitate and optimise the various components of the public health response, particularly vaccination. Lessons learned from our recent experience may inform risk communication approaches in other countries that may face a similar situation as global polio eradication moves towards the 'End game'.

  13. CpG oligodeoxynucleotides are a potent adjuvant for an inactivated polio vaccine produced from Sabin strains of poliovirus.

    PubMed

    Yang, Chunting; Shi, Huiying; Zhou, Jun; Liang, Yanwen; Xu, Honglin

    2009-11-05

    Poliovirus transmission is controlled globally through world-wide use of a live attenuated oral polio vaccine (OPV). However, the imminence of global poliovirus eradication calls for a switch to the inactivated polio vaccine (IPV). Given the limited manufacturing capacity and high cost of IPV, this switch is unlikely in most developing and undeveloped countries. Adjuvantation is an effective strategy for antigen sparing. In this study, we evaluated the adjuvanticity of CpG oligodeoxynucleotides (CpG-ODN) for an experimental IPV produced from Sabin strains of poliovirus. Our results showed that CpG-ODN, alone or in combination with alum, can significantly enhance both the humoral and cellular immune responses to IPV in mice, and, consequently, the antigen dose could be reduced substantially. Therefore, our study suggests that the global use of IPV could be facilitated by using CpG-ODN or other feasible adjuvants.

  14. Hybrid proteins between Pseudomonas aeruginosa exotoxin A and poliovirus 2Apro cleave p220 in HeLa cells.

    PubMed Central

    Novoa, I; Cotten, M; Carrasco, L

    1996-01-01

    Cleavage of p220, a component of the initiation factor eIF-4F, has been correlated with the inhibition of host translation during poliovirus infection. To obtain p220 cleavage in the absence of any other poliovirus gene products, hybrid proteins containing Pseudomonas aeruginosa exotoxin A and poliovirus protease 2Apro have been constructed. The addition of the hybrid molecules to cultured cells did not lead to substantial p220 cleavage. However, the simultaneous presence of the hybrid toxin with replicationally inactive chicken adenovirus particles results in efficient cleavage of p220 in the intact cells. Under these conditions, cellular translation continues unabated for several hours, arguing against a direct requirement for intact p220 in each round of the initiation of translation of cellular mRNAs. PMID:8627818

  15. Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus.

    PubMed

    Aldabe, R; Feduchi, E; Novoa, I; Carrasco, L

    1995-10-24

    Poliovirus protease 2A cleaves p220, a component of initiation factor eIF-4F. Polyclonal antibodies that recognize p220 and the cleaved products from different species have been raised. Transfection of several cell lines with poliovirus 2Apro cloned in different plasmids leads to efficient cleavage of p220 upon infection with VT7, a recombinant vaccinia virus that expresses the T7 RNA polymerase. Under these conditions vaccinia virus protein synthesis is severely inhibited, while expression of poliovirus protein 2C from a similar plasmid has no effect. These results show by the first time the effects of p220 cleavage on vaccinia virus translation in the infected cells.

  16. Acute flaccid paralysis surveillance in bosnia and herzegovina: Recent isolation of two sabin like type 2 poliovirus.

    PubMed

    Fontana, Stefano; Buttinelli, Gabriele; Fiore, Stefano; Mulaomerovic, Mirsada; Aćimović, Jela; Amato, Concetta; Delogu, Roberto; Rezza, Giovanni; Stefanelli, Paola

    2017-09-01

    The WHO Regional Commission for the Certification of Poliomyelitis Eradication has recently indicated Bosnia and Herzegovina (B&H) as a high risk country for transmission, following importation, of wild poliovirus (WPV) or circulating vaccine-derived poliovirus (cVDPV). We analyzed data on Acute Flaccid Paralysis (AFP) surveillance between 2007 to 2016, and the trend of polio immunization coverage in B&H. The majority of AFP cases was recorded in 2016 suggesting an enhancement of the AFP surveillance activities. However, the decline in the immunization coverage, around 74%, and the isolation of two Sabin-like poliovirus type 2 strains, one of them close to a VDPV, require a particular attention in the area. Although B&H has successfully maintained its polio-free status since 2002 several challenges need to be addressed. © 2017 Wiley Periodicals, Inc.

  17. VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

    PubMed Central

    Lin, Kai; Perni, Robert B.; Kwong, Ann D.; Lin, Chao

    2006-01-01

    The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells. PMID:16641454

  18. Extreme Susceptibility of African Naked Mole Rats (Heterocephalus glaber) to Experimental Infection with Herpes Simplex Virus Type 1

    PubMed Central

    Artwohl, James; Ball-Kell, Susan; Valyi-Nagy, Tibor; Wilson, Steven P; Lu, Ying; Park, Thomas J

    2009-01-01

    Herpes simplex virus type 1 (HSV1) is widely used as a gene delivery vector in a variety of laboratory animals. In a recent study, a thymidine-kinase–inactive (replication-conditional) HSV1 used as a delivery vector was lethal in naked mole rats, whereas mice infected with the identical virus showed no adverse effects. This result prompted us to undertake a controlled comparative histologic study of the effect of HSV1 infection on naked mole rats and mice. Replication-competent and replication-conditional HSV1 caused widespread inflammation and necrosis in multiple organ systems of naked mole rats but not mice; naked mole rats infected with replication-defective virus showed no adverse effects. We conclude that the lethality of HSV1 for naked mole rats is likely the result of overwhelming infection, possibly in part due to this species’ natural lack of proinflammatory neuropeptides at the initial site of infection. PMID:19295058

  19. Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative

    PubMed Central

    Dunn, Glynis; Klapsa, Dimitra; Wilton, Thomas; Stone, Lindsay; Minor, Philip D.; Martin, Javier

    2015-01-01

    There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era. PMID:26313548

  20. Identification of an alternative open reading frame ("hidden gene"?) stringently required for infectivity of poliovirus cDNA clones.

    PubMed

    Pierangeli, A; Bucci, M; Forzan, M; Pagnotti, P; Equestre, M; Pérez Bercoff, R

    1998-10-01

    Translation of the uncapped poliovirus RNA starts at the AUG triplet spanning positions 741-743, and proceeds uninterrupted for almost the entire length of the genome. Such a cap-independent mechanism of internal initiation of translation determines that a long, extra-cistronic region extends between the 5'-end and the main open reading frame (ORF). We have identified 10 short ORFs initiated by the alternative translation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-cistronic region (5'-ECR) of poliovirus RNA. Mutations introduced in all but one of these mini-cistrons had no effect on the infectivity of full-length cDNA clones, except when they modified a "hidden frame" spanning between nucleotides 157-192 (starting triplet: ACG). The mini cistron 157-192 is conserved in position, length and sequence in the genome of all types and strains of poliovirus. Adaptation to rat (Lansing) or mouse (variant of Sabin 2) is accompanied by a consistent pattern of changes in the primary sequence of this "hidden frame". The substitutions that abrogated the infectivity of cDNA clones were not expected to modify the predicted secondary structure of the 5'-ECR, and they did not alter the ability of the IRES to direct internal initiation of translation in bi-cistronic mRNAs. The infectivity of the mutated poliovirus cDNAs could be complemented in trans by co-transfecting the target COS-1 cells with an expression vector containing just the 5'-ECR of poliovirus type 2 (Lansing strain). The infectivity of poliovirus cDNA could be restored by co-transfecting short RNA transcripts of the wt 5'-ECR (Lansing), suggesting that the complementation in trans indeed requires the expression of the helper cDNA clone.

  1. Multiple Independent Emergences of Type 2 Vaccine-Derived Polioviruses during a Large Outbreak in Northern Nigeria

    PubMed Central

    Shaw, Jing; Jorba, Jaume; Bukbuk, David; Adu, Festus; Gumede, Nicksy; Pate, Muhammed Ali; Abanida, Emmanuel Ade; Gasasira, Alex; Iber, Jane; Chen, Qi; Vincent, Annelet; Chenoweth, Paul; Henderson, Elizabeth; Wannemuehler, Kathleen; Naeem, Asif; Umami, Rifqiyah Nur; Nishimura, Yorihiro; Shimizu, Hiroyuki; Baba, Marycelin; Adeniji, Adekunle; Williams, A. J.; Kilpatrick, David R.; Oberste, M. Steven; Wassilak, Steven G.; Tomori, Oyewale; Pallansch, Mark A.; Kew, Olen

    2013-01-01

    Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5′-untranslated region [5′-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5′-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries. PMID:23408630

  2. Twenty-Eight Years of Poliovirus Replication in an Immunodeficient Individual: Impact on the Global Polio Eradication Initiative.

    PubMed

    Dunn, Glynis; Klapsa, Dimitra; Wilton, Thomas; Stone, Lindsay; Minor, Philip D; Martin, Javier

    2015-08-01

    There are currently huge efforts by the World Health Organization and partners to complete global polio eradication. With the significant decline in poliomyelitis cases due to wild poliovirus in recent years, rare cases related to the use of live-attenuated oral polio vaccine assume greater importance. Poliovirus strains in the oral vaccine are known to quickly revert to neurovirulent phenotype following replication in humans after immunisation. These strains can transmit from person to person leading to poliomyelitis outbreaks and can replicate for long periods of time in immunodeficient individuals leading to paralysis or chronic infection, with currently no effective treatment to stop excretion from these patients. Here, we describe an individual who has been excreting type 2 vaccine-derived poliovirus for twenty eight years as estimated by the molecular clock established with VP1 capsid gene nucleotide sequences of serial isolates. This represents by far the longest period of excretion described from such a patient who is the only identified individual known to be excreting highly evolved vaccine-derived poliovirus at present. Using a range of in vivo and in vitro assays we show that the viruses are very virulent, antigenically drifted and excreted at high titre suggesting that such chronic excreters pose an obvious risk to the eradication programme. Our results in virus neutralization assays with human sera and immunisation-challenge experiments using transgenic mice expressing the human poliovirus receptor indicate that while maintaining high immunisation coverage will likely confer protection against paralytic disease caused by these viruses, significant changes in immunisation strategies might be required to effectively stop their occurrence and potential widespread transmission. Eventually, new stable live-attenuated polio vaccines with no risk of reversion might be required to respond to any poliovirus isolation in the post-eradication era.

  3. CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity.

    PubMed

    Liu, Chen; Zhu, Haizhen; Tu, Zhengkun; Xu, Yi-Ling; Nelson, David R

    2003-06-01

    The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.

  4. In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons.

    PubMed

    Graff, Judith; Nguyen, Hanh; Kasorndorkbua, Chaiyan; Halbur, Patrick G; St Claire, Marisa; Purcell, Robert H; Emerson, Suzanne U

    2005-01-01

    Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

  5. A Template RNA Entry Channel in the Fingers Domain of the Poliovirus Polymerase

    PubMed Central

    Kortus, Matthew G.; Kempf, Brian J.; Haworth, Kevin G.; Barton, David J.; Peersen, Olve B.

    2012-01-01

    Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase (3Dpol) that is required for viral RNA replication. Structures of 3Dpol from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a xseries of alanine substitution mutation sat these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for elongation complex stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3Dpol pinky finger drastically decreased the rate of initiation as well as elongation complex stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or elongation complex stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA elongation complexes. PMID:22321798

  6. Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

    SciTech Connect

    Takegami, T.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.

  7. Mutational analysis of poliovirus 2Apro. Distinct inhibitory functions of 2apro on translation and transcription.

    PubMed

    Ventoso, I; Barco, A; Carrasco, L

    1998-10-23

    Transient expression of poliovirus 2Apro in mammalian cells by means of the recombinant vaccinia virus vT7 expression system leads to drastic inhibition of both cellular and vaccinia virus gene expression (Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) FEBS Lett. 377, 1-5; Aldabe, R., Feduchi, E., Novoa, I., and Carrasco, L. (1995) Biochem. Biophys. Res. Commun. 215, 928-936). To obtain further insights into the molecular basis of this inhibition, a number of 2Apro variants were generated and expressed in COS-1 cells. The effect of these variants on cellular translation, on vaccinia virus-specific translation, and on transcription of the reporter gene luciferase was analyzed. The ability of the different 2Apro variants to block cellular translation depends on their capacities to cleave eIF-4G. The blockade exerted by 2Apro on transcription of the luciferase gene reinforces the notion that this protease is a potent inhibitor of RNA polymerase II-mediated transcription. Some of the 2Apro variants tested failed to block luciferase transcription, despite the fact that eIF-4G cleavage and inhibition of translation were observed. Two reconstituted polioviruses mutated in 2Apro were defective in inhibiting luciferase transcription, yet were still able to cleave eIF-4G and block translation. These findings indicate that 2Apro interferes with cellular gene expression at both the transcriptional and translational levels. Moreover, these two effects probably reflect the inactivation of different host proteins by poliovirus 2Apro.

  8. Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases.

    PubMed Central

    Nicklin, M J; Kräusslich, H G; Toyoda, H; Dunn, J J; Wimmer, E

    1987-01-01

    Plasmids have been constructed to generate substrates for the study of proteinases 2A and 3C of poliovirus. They contain the P1 (capsomer precursor) region of the poliovirus genome or P1 and part of P2 (a nonstructural precursor), which can be transcribed and translated in vitro. A transcript containing the entire 5' nontranslated region and the P1 region of the viral RNA gave poor translation in a reticulocyte translation system. Truncation of the 5' nontranslated region to its 3'-most segment gave acceptably good yields of radiolabeled P1. P1 was specifically processed to yield capsomer proteins by enzymes supplied in a postmitochondrial supernatant from poliovirus-infected cells. Thus, proteinase 3C can be supplied exogenously (in trans) and effect processing. This system may be used to provide P1 for the assay of proteinase 3C. Precursors that lacked either the 1A or 1D regions were poor substrates for proteinase 3C--observations that demonstrated a stringent structural requirement in processing by 3C. The translation product of a transcript encoding P1 and part of P2 was rapidly cleaved at the P1-P2 site in the absence of infected-cell extract. A transcript that contained a mutated 2A region gave a stable P1-P2 precursor that could be processed specifically by exogenous proteinase from infected-cell fractions. Processing of P1 appeared to require cleavage of the P1-P2 bond. These results support our previous data that 2A is the second polioviral proteinase and also provides a means of assaying proteinase 2A in vitro. Images PMID:3035560

  9. Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

    PubMed Central

    Kirkegaard, K; Nelsen, B

    1990-01-01

    Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

  10. Unraveling the message: insights into comparative genomics of the naked mole-rat.

    PubMed

    Lewis, Kaitlyn N; Soifer, Ilya; Melamud, Eugene; Roy, Margaret; McIsaac, R Scott; Hibbs, Matthew; Buffenstein, Rochelle

    2016-08-01

    Animals have evolved to survive, and even thrive, in different environments. Genetic adaptations may have indirectly created phenotypes that also resulted in a longer lifespan. One example of this phenomenon is the preternaturally long-lived naked mole-rat. This strictly subterranean rodent tolerates hypoxia, hypercapnia, and soil-based toxins. Naked mole-rats also exhibit pronounced resistance to cancer and an attenuated decline of many physiological characteristics that often decline as mammals age. Elucidating mechanisms that give rise to their unique phenotypes will lead to better understanding of subterranean ecophysiology and biology of aging. Comparative genomics could be a useful tool in this regard. Since the publication of a naked mole-rat genome assembly in 2011, analyses of genomic and transcriptomic data have enabled a clearer understanding of mole-rat evolutionary history and suggested molecular pathways (e.g., NRF2-signaling activation and DNA damage repair mechanisms) that may explain the extraordinarily longevity and unique health traits of this species. However, careful scrutiny and re-analysis suggest that some identified features result from incorrect or imprecise annotation and assembly of the naked mole-rat genome: in addition, some of these conclusions (e.g., genes involved in cancer resistance and hairlessness) are rejected when the analysis includes additional, more closely related species. We describe how the combination of better study design, improved genomic sequencing techniques, and new bioinformatic and data analytical tools will improve comparative genomics and ultimately bridge the gap between traditional model and nonmodel organisms.

  11. Development of "Naked-Tufted" Seed Coat Mutants for Potential Use in Cotton Production

    USDA-ARS?s Scientific Manuscript database

    Use of chemical mutagenesis has been highly successful in most major crops but has only recently been used in improving cotton. The objective of this research was to develop ‘naked-tufted’ seed mutants and to incorporate this genetic trait into cotton to enhance crop quality and reduce processing co...

  12. Blunted behavioral and c Fos responses to acidic fumes in the African naked mole-rat.

    PubMed

    LaVinka, Pamela Colleen; Park, Thomas J

    2012-01-01

    Acidosis in the skin triggers activation of pain pathways and behaviors indicative of pain in vertebrates. The exception is the naked mole-rat, the only known vertebrate to show physiological and behavioral insensitivity to acid pain in the skin. The goal of the present study was to determine behavioral and physiological responses of this species to airborne acidic fumes, which would be expected to affect the trigeminal pain pathway in other species. Behaviorally, naked mole-rats did not avoid fumes from moderately high concentrations of acetic acid (10 and 20%), and c Fos labeling showed no increase in activity in the trigeminal nuclei and nucleus tractus solitarius. In contrast, these concentrations triggered behavioral aversion and increased Fos activity in other laboratory rodents. For a very high concentration of acetic acid (50%), naked mole-rats showed significant avoidance behavior and increased Fos labeling in the nucleus tractus solitarius caudal region, which receives vagal chemosensory information. However, there was no increase in trigeminal labeling, and in fact, activity significantly decreased. This pattern is opposite of that associated with another irritant, ammonia fumes, which elicited an increase in trigeminal but not nucleus tractus solitarius Fos labeling, and no behavioral avoidance. Behavioral avoidance of acidic fumes, but no increased labeling in the trigeminal pain nucleus is consistent with the notion of adaptations to blunt acid pain, which would be advantageous for naked mole-rats as they normally live under chronically high levels of acidosis-inducing CO(2).

  13. Naked eye detection of nitric oxide release from nitrosothiols aided by gold nanoparticles.

    PubMed

    Priya, S; Kaviyarasan, T; Berchmans, Sheela

    2012-04-07

    In this work we have demonstrated that nitric oxide can be monitored spectrophotometrically using cyclodextrin encapsulated ferrocene. The detection course showed the colour change from yellow to blue which can be detected with the naked eye. Also we describe the catalytic effect of gold nanoparticles in enhancing nitric oxide release from S-nitrosothiols.

  14. Intrathecal Injection of Naked Plasmid DNA Provides Long-term Expression of Secreted Proteins

    PubMed Central

    Hughes, Travis S; Langer, Stephen J; Johnson, Kirk W; Chavez, Raymond A; Watkins, Linda R; Milligan, Erin D; Leinwand, Leslie A

    2008-01-01

    Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (~vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF. PMID:18941439

  15. Registration of tufted-naked seed in upland cotton germplasm 9023n4t

    USDA-ARS?s Scientific Manuscript database

    A naked-tufted mutant called 9023n4t (PI 667553) was developed from the cultivar SC 9023 (Gossypium hirsutum L.) through chemical mutagenesis. This germplasm was developed by the Department of Plant and Soil Science, Texas Tech University and released in April, 2013. This mutant is quite unique sinc...

  16. Blunted Behavioral and C Fos Responses to Acidic Fumes in the African Naked Mole-Rat

    PubMed Central

    LaVinka, Pamela Colleen; Park, Thomas J.

    2012-01-01

    Acidosis in the skin triggers activation of pain pathways and behaviors indicative of pain in vertebrates. The exception is the naked mole-rat, the only known vertebrate to show physiological and behavioral insensitivity to acid pain in the skin. The goal of the present study was to determine behavioral and physiological responses of this species to airborne acidic fumes, which would be expected to affect the trigeminal pain pathway in other species. Behaviorally, naked mole-rats did not avoid fumes from moderately high concentrations of acetic acid (10 and 20%), and c Fos labeling showed no increase in activity in the trigeminal nuclei and nucleus tractus solitarius. In contrast, these concentrations triggered behavioral aversion and increased Fos activity in other laboratory rodents. For a very high concentration of acetic acid (50%), naked mole-rats showed significant avoidance behavior and increased Fos labeling in the nucleus tractus solitarius caudal region, which receives vagal chemosensory information. However, there was no increase in trigeminal labeling, and in fact, activity significantly decreased. This pattern is opposite of that associated with another irritant, ammonia fumes, which elicited an increase in trigeminal but not nucleus tractus solitarius Fos labeling, and no behavioral avoidance. Behavioral avoidance of acidic fumes, but no increased labeling in the trigeminal pain nucleus is consistent with the notion of adaptations to blunt acid pain, which would be advantageous for naked mole-rats as they normally live under chronically high levels of acidosis-inducing CO2. PMID:23028761

  17. Intrathecal injection of naked plasmid DNA provides long-term expression of secreted proteins.

    PubMed

    Hughes, Travis S; Langer, Stephen J; Johnson, Kirk W; Chavez, Raymond A; Watkins, Linda R; Milligan, Erin D; Leinwand, Leslie A

    2009-01-01

    Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.

  18. A luminescent heterometallic metal-organic framework for the naked-eye discrimination of nitroaromatic explosives.

    PubMed

    Qi, Xiujuan; Jin, Yunhe; Li, Na; Wang, Zhi; Wang, Kangcai; Zhang, Qinghua

    2017-09-05

    A luminescent heterometallic MOF with an interrupted cap topology was synthesized and characterized. This compound exhibits a strong blue-light emission and shows naked-eye selective detection toward trinitrobenzene (TNB) even in the presence of other nitro explosives. Furthermore, the compound is also capable of selectively sensing nitroaromatic picric acid (PA) based on the fluorescence quenching mechanism.

  19. Virtually naked: virtual environment reveals sex-dependent nature of skin disclosure.

    PubMed

    Lomanowska, Anna M; Guitton, Matthieu J

    2012-01-01

    The human tendency to reveal or cover naked skin reflects a competition between the individual propensity for social interactions related to sexual appeal and interpersonal touch versus climatic, environmental, physical, and cultural constraints. However, due to the ubiquitous nature of these constraints, isolating on a large scale the spontaneous human tendency to reveal naked skin has remained impossible. Using the online 3-dimensional virtual world of Second Life, we examined spontaneous human skin-covering behavior unhindered by real-world climatic, environmental, and physical variables. Analysis of hundreds of avatars revealed that virtual females disclose substantially more naked skin than virtual males. This phenomenon was not related to avatar hypersexualization as evaluated by measurement of sexually dimorphic body proportions. Furthermore, analysis of skin-covering behavior of a population of culturally homogeneous avatars indicated that the propensity of female avatars to reveal naked skin persisted despite explicit cultural norms promoting less revealing attire. These findings have implications for further understanding how sex-specific aspects of skin disclosure influence human social interactions in both virtual and real settings.

  20. Virtually Naked: Virtual Environment Reveals Sex-Dependent Nature of Skin Disclosure

    PubMed Central

    Lomanowska, Anna M.; Guitton, Matthieu J.

    2012-01-01

    The human tendency to reveal or cover naked skin reflects a competition between the individual propensity for social interactions related to sexual appeal and interpersonal touch versus climatic, environmental, physical, and cultural constraints. However, due to the ubiquitous nature of these constraints, isolating on a large scale the spontaneous human tendency to reveal naked skin has remained impossible. Using the online 3-dimensional virtual world of Second Life, we examined spontaneous human skin-covering behavior unhindered by real-world climatic, environmental, and physical variables. Analysis of hundreds of avatars revealed that virtual females disclose substantially more naked skin than virtual males. This phenomenon was not related to avatar hypersexualization as evaluated by measurement of sexually dimorphic body proportions. Furthermore, analysis of skin-covering behavior of a population of culturally homogeneous avatars indicated that the propensity of female avatars to reveal naked skin persisted despite explicit cultural norms promoting less revealing attire. These findings have implications for further understanding how sex-specific aspects of skin disclosure influence human social interactions in both virtual and real settings. PMID:23300580

  1. Newtonian analogue of static general relativistic spacetimes: An extension to naked singularities

    NASA Astrophysics Data System (ADS)

    Ghosh, Shubhrangshu; Sarkar, Tamal; Bhadra, Arunava

    2015-10-01

    We formulate a generic Newtonian-like analogous potential for static spherically symmetric general relativistic (GR) spacetime and subsequently derived proper Newtonian-like analogous potential corresponding to Janis-Newman-Winicour (JNW) and Reissner-Nordström (RN) spacetimes, both exhibiting naked singularities. The derived potentials were found to reproduce the entire GR features including the orbital dynamics of the test particle motion and the orbital trajectories, with precise accuracy. The nature of the particle orbital dynamics including their trajectory profiles in JNW and RN geometries show altogether different behaviors with distinctive traits as compared to the nature of particle dynamics in Schwarzschild geometry. Exploiting the Newtonian-like analogous potentials, we found that the radiative efficiency of a geometrically thin and optically thick Keplerian accretion disk around naked singularities corresponding to both JNW and RN geometries, in general, is always higher than that for Schwarzschild geometry. The derived potentials would thus be useful to study astrophysical processes, especially to investigate more complex accretion phenomena in active galactic nuclei (AGNs) or in x-ray binaries (XRBs) in the presence of naked singularities and thereby to explore any noticeable differences in their observational features from those in the presence of black holes (BHs) to ascertain outstanding debatable issues relating to gravity—whether the end state of gravitational collapse in our physical Universe renders BH or naked singularity.

  2. The combined use of alphavirus replicons and pseudoinfectious particles for the discovery of antivirals derived from natural products.

    PubMed

    Delekta, Phillip C; Raveh, Avi; Larsen, Martha J; Schultz, Pamela J; Tamayo-Castillo, Giselle; Sherman, David H; Miller, David J

    2015-06-01

    Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.

  3. Update on Vaccine-Derived Polioviruses - Worldwide, January 2014-March 2015.

    PubMed

    Diop, Ousmane M; Burns, Cara C; Sutter, Roland W; Wassilak, Steven G; Kew, Olen M

    2015-06-19

    Since the World Health Assembly's 1988 resolution to eradicate poliomyelitis, one of the main tools of the World Health Organization (WHO) Global Polio Eradication Initiative (GPEI) has been the live, attenuated oral poliovirus vaccine (OPV). OPV might require several doses to induce immunity but provides long-term protection against paralytic disease. Through effective use of OPV, GPEI has brought polio to the threshold of eradication. Wild poliovirus type 2 (WPV2) was eliminated in 1999, WPV3 has not been detected since November 2012, and WPV1 circulation appears to be restricted to parts of Pakistan and Afghanistan. However, continued use of OPV carries two key risks. The first, vaccine-associated paralytic poliomyelitis (VAPP) has been recognized since the early 1960s. VAPP is a very rare event that occurs sporadically when an administered dose of OPV reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can occur among immunologically normal vaccine recipients and their contacts as well as among persons who have primary immunodeficiencies (PIDs) manifested by defects in antibody production; it is not associated with outbreaks. The second, the emergence of genetically divergent, neurovirulent vaccine-derived polioviruses (VDPVs) was recognized more recently. Circulating VDPVs (cVDPVs) resemble WPVs and, in areas with low OPV coverage, can cause polio outbreaks. Immunodeficiency-associated VDPVs (iVDPVs) can replicate and be excreted for years in some persons with PIDs; GPEI maintains a registry of iVDPV cases. Ambiguous VDPVs (aVDPVs) are isolates that cannot be classified definitively. This report updates previous surveillance summaries and describes VDPVs detected worldwide during January 2014-March 2015. Those include new cVDPV outbreaks in Madagascar and South Sudan, and sharply reduced type 2 cVDPV (cVDPV2) circulation in Nigeria and Pakistan during the latter half of 2014. Eight newly identified persons in

  4. Modified procedure for the recovery of naturally accumulated poliovirus from oysters.

    PubMed Central

    Vaughn, J M; Landry, E F; Vicale, T J; Dahl, M C

    1979-01-01

    Methods were compared for their ability to recover poliovirus from oysters (Crassostrea gigas) which had been allowed to accumulate virus via normal filtration activities. Clarification procedures included glycine-NaCl and polyelectrolyte extraction methods followed by a variety of acid precipitation concentration methods. Polyelectrolyte flocculation followed by a beef extract-supplemented acid precipitation carried out at pH 3.5 yielded the most efficient recoveries. Direct assay of homogenates was found to be an unreliable method for determining the initial virus concentration in "naturally infected" oysters. PMID:231934

  5. Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis▿

    PubMed Central

    Brisac, Cynthia; Téoulé, François; Autret, Arnaud; Pelletier, Isabelle; Colbère-Garapin, Florence; Brenner, Catherine; Lemaire, Christophe; Blondel, Bruno

    2010-01-01

    We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca2+) concentration in neuroblastoma IMR5 cells, at least partly through Ca2+ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) channels. This leads to Ca2+ accumulation in mitochondria through the mitochondrial Ca2+ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca2+ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis. PMID:20861253

  6. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion.

    PubMed

    Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

    2015-10-01

    The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1-147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1-147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex.

  7. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion

    PubMed Central

    Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

    2015-01-01

    The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1–147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1–147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex. PMID

  8. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles.

    PubMed

    McCullough, Kenneth C; Milona, Panagiota; Thomann-Harwood, Lisa; Démoulins, Thomas; Englezou, Pavlos; Suter, Rolf; Ruggli, Nicolas

    2014-10-16

    Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

  9. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    PubMed Central

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  10. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

    PubMed Central

    Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S.; Chen, Qiang

    2011-01-01

    Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing. PMID:21883868

  11. Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon.

    PubMed

    Alvarez-Martín, Pablo; Belén Flórez, Ana; Margolles, Abelardo; del Solar, Gloria; Mayo, Baltasar

    2008-08-01

    This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

  12. Search for poliovirus carriers among people with primary immune deficiency diseases in the United States, Mexico, Brazil, and the United Kingdom.

    PubMed Central

    Halsey, Neal A.; Pinto, Jorge; Espinosa-Rosales, Francisco; Faure-Fontenla, María A.; da Silva, Edson; Khan, Aamir J.; Webster, A. D.; Minor, Philip; Dunn, Glynis; Asturias, Edwin; Hussain, Hamidah; Pallansch, Mark A.; Kew, Olen M.; Winkelstein, Jerry; Sutter, Roland

    2004-01-01

    OBJECTIVE: To estimate the rate of long-term poliovirus excretors in people known to have B-cell immune deficiency disorders. METHODS: An active search for chronic excretors was conducted among 306 persons known to have immunoglobulin G (IgG) deficiency in the United States, Mexico, Brazil, and the United Kingdom, and 40 people with IgA deficiency in the United States. Written informed consent or assent was obtained from the participants or their legal guardians, and the studies were formally approved. Stool samples were collected from participants and cultured for polioviruses. Calculation of the confidence interval for the proportion of participants with persistent poliovirus excretion was based on the binomial distribution. FINDINGS: No individuals with long-term excretion of polioviruses were identified. Most participants had received oral poliovirus vaccine (OPV) and almost all had been exposed to household contacts who had received OPV. Polioviruses of recent vaccine origin were transiently found in four individuals in Mexico and Brazil, where OPV is recommended for all children. CONCLUSION: Although chronic poliovirus excretion can occur in immunodeficient persons, it appears to be rare. PMID:15106294

  13. Screening for long-term poliovirus excretion among children with primary immunodeficiency disorders: preparation for the polio posteradication era in Bangladesh.

    PubMed

    Sazzad, Hossain M S; Rainey, Jeanette J; Kahn, Anna-Lea; Mach, Ondrej; Liyanage, Jayantha B L; Alam, Ahmed Nawsher; Kawser, Choudhury A; Hossain, Asgar; Sutter, Roland; Luby, Stephen P

    2014-11-01

    Persons with primary immune deficiency disorders (PIDD) who receive oral poliovirus vaccine (OPV) may transmit immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) and cause paralytic polio. The objective of this study was to identify children with PIDD in Bangladesh, and estimate the proportion with chronic poliovirus excretion. Patients admitted at 5 teaching hospitals were screened for PIDD according to standardized clinical case definitions. PIDD was confirmed by age-specific quantitative immunoglobulin levels. Stool specimens were collected from patients with confirmed PIDD. From February 2011 through January 2013, approximately 96 000 children were screened, and 53 patients were identified who met the clinical case definition for PIDD. Thirteen patients (24%) had age-specific quantitative immunoglobulins results that confirmed PIDD. Of these, 9 (69%) received OPV 3-106 months before stool specimen collection. Among 11 patients, stool specimens from 1 patient tested positive for polioviruses 34 months after OPV ingestion. However, the poliovirus isolate was not available for genetic sequencing, and a subsequent stool specimen 45 days later was negative. The risk of chronic poliovirus excretion among children with PIDD in Bangladesh seems to be low. The national polio eradication program should incorporate strategies for screening for poliovirus excretion among patients with PIDD. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residue at positions of codon degeneracy.

    PubMed Central

    Kilpatrick, D R; Nottay, B; Yang, C F; Yang, S J; Mulders, M N; Holloway, B P; Pallansch, M A; Kew, O M

    1996-01-01

    We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to approximately 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable. PMID:8940436

  15. Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells

    PubMed Central

    Mohanty, Madhu C.; Deshpande, Jagadish M.

    2013-01-01

    Background & objectives: Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains) do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV) and Sabin attenuated type 1 poliovirus (Sabin PV) in cultured human neuronal cells. Methods: By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs) and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH) infected with Sabin PV and wild PV. Results: Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5) m-RNA in neuronal cells at the beginning of infection (up to 4 h) as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Interpretation & conclusions: Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies. PMID:24056597

  16. An outbreak of wild poliovirus in the Republic of Congo, 2010-2011.

    PubMed

    Patel, Minal K; Konde, Mandy Kader; Didi-Ngossaki, Boris Hermann; Ndinga, Edouard; Yogolelo, Riziki; Salla, Mbaye; Shaba, Keith; Everts, Johannes; Armstrong, Gregory L; Daniels, Danni; Burns, Cara; Wassilak, Steve; Pallansch, Mark; Kretsinger, Katrina

    2012-11-15

    The Republic of Congo has had no cases of wild poliovirus type 1 (WPV1) since 2000. In October 2010, a neurologist noted an abnormal number of cases of acute flaccid paralysis (AFP) among adults, which were later confirmed to be caused by WPV1. Those presenting with AFP underwent clinical history, physical examination, and clinical specimen collection to determine if they had polio. AFP cases were classified as laboratory-confirmed, clinical, or nonpolio AFP. Epidemiologic features of the outbreak were analyzed. From 19 September 2010 to 22 January 2011, 445 cases of WPV1 were reported in the Republic of Congo; 390 cases were from Pointe Noire. Overall, 331 cases were among adults; 378 cases were clinically confirmed, and 64 cases were laboratory confirmed. The case-fatality ratio (CFR) was 43%. Epidemiologic characteristics differed among polio cases reported in Pointe Noire and cases reported in the rest of the Republic of Congo, including age distribution and CFR. The outbreak stopped after multiple vaccination rounds with oral poliovirus vaccine, which targeted the entire population. This outbreak underscores the need to maintain high vaccination coverage to prevent outbreaks, the need to maintain timely high-quality surveillance to rapidly identify and respond to any potential cases before an outbreak escalates, and the need to perform ongoing risk assessments of immunity gaps in polio-free countries.

  17. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    NASA Astrophysics Data System (ADS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  18. Poliovirus retention in 75-cm soil cores after sewage and rainwater application

    SciTech Connect

    Landry, E.F.; Vaughn, J.M.; Penello, W.F.

    1980-12-01

    The adsorption rate of a guanidine-resistant strain of poliovirus LSc 2ab was measured in Long Island soils with in situ field cores (10.1 by 75 cm). The test virus was chosen because it exhibited soil adsorption and elution characteristics of a number of non-polioviruses. After the inoculation of cores with seeded sewage effluent at a 1-cm/h infiltration rate, cores were extracted, fractionated, and analyzed for total plaque-forming units per each 5-cm fraction. The results showed that 77% of the viruses were adsorbed in the first 5 cm of soil. An additional 11% were found in the 5- to 10-cm fraction, and a total of 96% of the viruses were adsorbed by 25 cm. The remaining 4% were uniformly distributed over the next 50 cm of soil, with a minimum of 0.23% in each soil section. Few viruses (< 0.22%) were observed in core filtrates. Analysis of the viral distribution pattern in seeded cores, after an application of a single rinse of either sewage effluent or rainwater, indicated that large-scale viral mobilization was absent. However, localized areas of viral movement were noted in both of the rinsed cores, with the rainwater rinsed cores exhibiting more extensive movement. All mobilized viruses were resorbed at lower core depths.

  19. Direct measurement of the poliovirus RNA polymerase error frequency in vitro

    SciTech Connect

    Ward, C.D.; Stokes, M.A.M.; Flanegan, J.B. )

    1988-02-01

    The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high, depending on the specific reaction conditions. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg{sup 2+} (pH 7.0) to 7.0 mM Mg{sup 2+} (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.

  20. [Role of the National Poliovirus Laboratory for the Program of eradication and poliomyelitis surveillance].

    PubMed

    Trallero, Gloria; Cabrerizo, María; Avellón, Ana

    2013-01-01

    The Spanish acute flaccid paralysis surveillance network is coordinated by the National Poliovirus Laboratory (NPL), which, since 1998, carries out polioviruses (PV) and other enteroviruses detected characterization by cell culture and molecular techniques. A total of 110,725 (70046+40679) samples were studied between 1998-2012 and enteroviruses were detected in 8% of these. Among these enteroviruses 241 PV were characterized as PV Sabin-like, except samples belong to an imported poliomyelitis case, all of which were characterised as vaccine derived PV type 2. The NPL has carried out the serotyping and the intratypic differentiation of all the isolated PV in Spain of any syndrome. It is shown that wild PV has not circulated in our country during the 15 years studied and that has led to the signing of the Act of the "eradication of poliomyelitis in Spain" by WHO in 2001, and the /"certification of the eradication of wild PV free for European countries" on 21 June 2002. Currently only 3 countries have endemic transmission of wild PV (Pakistan, Afghanistan and Nigeria). Until a complete worldwide eradication, was achieved, Spain will actively continue to participate in the maintenance of the poliomyelitis eradication infrastructure by monitoring and vaccination as well as the wild PV containment plan to avoid the spread of wild PV.

  1. Genotype-specific RNA probes for direct identification of wild polioviruses by blot hybridization.

    PubMed Central

    De, L; Yang, C F; Da Silva, E; Boshell, J; Cáceres, P; Gómez, J R; Pallansch, M; Kew, O

    1997-01-01

    We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corr