DOCSLIB.ORG

Construction of Intertypic Chimeric Dengue Viruses by Substitution Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Construction of Intertypic Chimeric Dengue Viruses by Substitution Of Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10342-10346, November 1991 Medical Sciences Construction of intertypic chimeric dengue viruses by substitution of structural protein genes (chimera/viral proteins/neurovirulence) MICHAEL BRAY AND CHING-JUH LAI Molecular Viral Biology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Robert M. Chanock, August 28, 1991 ABSTRACT Dengue virus contains an li-kilobase posi- temperature sensitive, and/or (iii) were adapted to cell cul- tive-strand RNA genome that codes for, in one open reading tures derived from an unnatural host (i.e., host range mu- frame, three structural proteins (capsid, premembrane, and tants) have been selected and evaluated as candidates for envelope), followed by seven nonstructural proteins. The struc- inclusion in a live attenuated virus vaccine (6-8). However, tural protein genes ofa full-length cDNA done of type 4 dengue despite 25 years of such efforts, safe, effective dengue virus were replaced with the corresponding genes of dengue 1 vaccines are still not available for general use. or dengue 2 to create intertypic chimeric cDNA. The RNA There are four serotypes of dengue viruses (types 1-4), transcripts made from these templates were infectious when which are distinguishable by plaque reduction neutralization transfected into permissive cells in culture. Progeny ofchimeric using serotype-specific monoclonal antibodies (mAbs) and cDNA produced apparently authentic dengue 1 or dengue 2 by less specific tests using polyclonal sera (9, 10). The structural proteins, together with dengue 4 nonstructural existence of serotypes was first discovered during early proteins, and as a consequence exhibited type 1 or type 2 studies in human volunteers, which showed that infection serological specificity. Both of the chimeras ultimately grew to with one dengue serotype induced durable homotypic immu- the same titer as their type 1 or type 2 parent, but the type nity, whereas heterotypic immunity lasted only 3-6 months 2/type 4 chimera grew very slowly. This chimera also pro- (2). It has been suggested that an effective dengue vaccine duced small plaques; in contrast, the type 1/type 4 chimera should contain all four serotypes in order to induce broad produced normal size plaques. The type 2/type 4 chimera immunity that would preclude the occurrence of DHF/DSS. retained the mouse neurovirulence ofthe dengue 2 virus, which The complete nucleotide sequences have been determined was the source of its structural protein genes. Each of the mice for dengue virus types 3 and 4 and several strains of type 2 inoculated intracerebrally with the chimera died, but survival virus, including the mouse-neurovirulent New Guinea C time was prolonged. The retardation of replication of the type (NGC) strain; however, only the 5' portion ofthe type 1 virus 2/type 4 chimeric virus suggests that this virus and possibly genome has been sequenced (11-16). The results of these other intertypic dengue virus chimeras with similar properties studies indicate that the four dengue virus serotypes share a should be examined for attenuation in primates and possible common genome organization. The genome of the dengue usefulness in a live dengue virus vaccine for humans. type 4 Caribbean strain 814669 was found to contain 10,646 nucleotides (11, 12). The first 101 nucleotides at the 5' end Dengue is a mosquito-borne viral disease that occurs in and the last 384 at the 3' end are noncoding. The remaining tropical and subtropical regions throughout the world. The sequence codes for a 3386 amino acid polyprotein, which dengue virus subgroup causes more human disease than any includes the three structural capsid (C), other member of the flavivirus family. Dengue is character- proteins-namely, ized by fever, rash, severe headache, and joint pain. Its pre-membrane (pre-M), and envelope (E), at its N terminus- mortality rate is low. However, over the past few decades, a followed by seven nonstructural proteins in the order NS1- more severe form of dengue, characterized by hemorrhage NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is and shock (dengue hemorrhagic fever/dengue shock syn- processed by cell signal peptidase(s) and by viral proteases to drome, or DHF/DSS) has been observed with increasing generate 11 or more viral proteins (17-20). frequency in children and young adults. DHF/DSS occurs We recently succeeded in cloning a stable, full-length most often during dengue virus infection of individuals pre- cDNA copy of type 4 dengue virus (designated p2A), which viously infected with another dengue virus serotype, and this can serve as a template for in vitro transcription of infectious has led to the suggestion that immune enhancement of viral RNA (21). Thus, it is now possible to introduce specific replication plays a role in pathogenesis of the more severe mutations into a full-length dengue cDNA and recover viable form of the disease (1). dengue viruses bearing those mutations. Mutants produced in Soon after their isolation in 1944, dengue viruses were this manner should be useful for biologic studies and for passaged repeatedly in mouse brain, resulting in the selection construction of live virus vaccines. We now report the of mouse neurovirulent mutants (2). Interestingly, studies construction of two viable chimeric dengue viruses through performed in volunteers showed that mouse brain-adapted replacement of the structural protein genes of the dengue neurovirulent mutants ofthree strains oftype 1 or type 2 virus type 4 cDNA clone with the corresponding genes oftype 1 or were attenuated, but still immunogenic for humans (2-5). The type 2 dengue virus. These chimeric viruses exhibit the mutants were not developed further as candidate vaccine serologic specificity of type 1 or type 2 but retain the strains because of concern for mouse brain antigens in the nonstructural proteins of type 4. The chimeric type 2/type 4 vaccine preparations. Since that time, virus mutants that (i) exhibited the small plaque size phenotype, and/or (ii) were Abbreviations: DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; pfu, plaque-forming unit(s); E protein, envelope protein; C protein, capsid protein; pre-M protein, pre-membrane protein; The publication costs of this article were defrayed in part by page charge IFA, immunofluorescence assay; HMAF, hyperimmune mouse as- payment. This article must therefore be hereby marked "advertisement" citic fluid; WP strain, Western Pacific strain; NGC strain, New in accordance with 18 U.S.C. §1734 solely to indicate this fact. Guinea C strain; mAb, monoclonal antibody. 10342 Downloaded by guest on September 30, 2021 Medical Sciences: Bray and Lai Proc. Natl. Acad. Sci. USA 88 (1991) 10343 virus retains the neurovirulence phenotype of dengue 2 NGC I-Xho I fragment was used to replace the corresponding strain, from which its structural genes were derived. fragment of p2A(Xho I) to create p2A(D2 NGC). RNA Transcription, Transfection, and Recovery of Virus. MATERIALS AND METHODS Transfection of cells with full-length RNA transcripts was performed as described (22). Ten days after transfection, Viruses. Dengue type 4 virus strain 814669 was used for cells were trypsinized and transferred to a 6-well plate and to construction offull-length cDNA that served as the source of a chamber slide. Two days later, cells on the slide were tested full-length infectious RNA transcripts (11, 12). A preparation by an immunofluorescence assay (IFA) for evidence of of dengue type 1 virus Western Pacific (WP) strain (D1 WP), dengue viral antigens. If IFA showed that most cells were fetal rhesus lung cell passage level 9, was kindly provided by infected, the cells in the 6-well plate were trypsinized, mixed K. Eckels (Walter Reed Army Institute of Research, Wash- with a 6-fold excess of uninfected cells, and incubated until ington) (21). A mouse brain preparation of mouse neurovir- cytopathic effects (appearance of numerous dead cells in the ulent dengue type 2 virus New Guinea C (NGC) strain (D2 medium) became evident, usually after 6 or 7 days. The NGC), mouse brain passage level 38, was kindly provided by infected cells were then harvested by removing the medium, D. Dubois (Walter Reed Army Institute of Research) (2). scraping the cells, resuspending them in a standard volume of Dengue 1 and dengue 2 viruses were amplified by one passage 50% Eagle's minimal essential medium/50%o serum, and then in C6/36 mosquito cells. Each ofthese three viruses was then freezing. If a small percentage of cells was positive, the cells passaged once in LLC-MK2 simian kidney cells, and the were in and to resulting virus suspension was used to study plaque mor- trypsinized, diluted 1:3 fresh medium, allowed phology and mouse neurovirulence. grow without addition of uninfected cells. The percentage of Cloning Vectors. Plasmid p5'-2, which contains the 5' half infected cells was then estimated on a weekly basis, and cell of the dengue 4 genome cDNA, was modified to facilitate lysates were harvested and titered for virus at intervals. replacement of the three structural protein genes (22). First, Detection of Dengue Virus Antigens. Infected cells were a unique Xho I site was introduced through site-directed analyzed by IFA using serotype-specific mAbs iF1 (dengue mutagenesis at nucleotide 2342 (A -+ G) of the dengue 4 1), 3H5 (dengue 2), 5D4 (dengue 3), and 1H10 (dengue 4); sequence, near the site encoding the C terminus of E protein, nonstructural protein NS1-specific mAb 1G6 (dengue 4), creating p5'-2(Xho I). This nucleotide change did not alter the originally produced by M. K. Gentry and E. Henchal (10); amino acid sequence. This vector was then digested at the and fluorescein-conjugated anti-mouse antiserum (Kierke- unique BstBI site within the dengue sequence and at the gaard & Perry Laboratories, Gaithersburg, MD).
Load more

Recommended publications
  • Patentability of Human-Animal Chimeras Ryan Hagglund
    Santa Clara High Technology Law Journal Volume 25 | Issue 1 Article 4 2008 Patentability of Human-Animal Chimeras Ryan Hagglund Follow this and additional works at: http://digitalcommons.law.scu.edu/chtlj Part of the Law Commons Recommended Citation Ryan Hagglund, Patentability of Human-Animal Chimeras, 25 Santa Clara High Tech. L.J. 51 (2012). Available at: http://digitalcommons.law.scu.edu/chtlj/vol25/iss1/4 This Article is brought to you for free and open access by the Journals at Santa Clara Law Digital Commons. It has been accepted for inclusion in Santa Clara High Technology Law Journal by an authorized administrator of Santa Clara Law Digital Commons. For more information, please contact [email protected]. PATENTABILITY OF HUMAN-ANIMAL CHIMERAS Ryan Hagglundt Abstract The chimera was a mythological creature with a lion 's head, goat's body, and serpent's tail. Because of recent advances in biotechnology, such permutations on species are no longer the stuff of myth and legend The term "chimera " has come to describe a class of genetically engineered creatures composed of some cells from one species, which thus contain genetic material derived entirely from that species, and some cells from another species, containing only genetic material from that species. Scientists have created a goat- sheep chimera or "geep" which exhibits physical characteristicsof both animals. Likewise, scientists have also used the tools of modern molecular biology to create human-animal chimeras containing both human and animal cells. While none of the human-animal chimeras hitherto created have exhibited significant human characteristics,the synthesis of human-animal chimeras raises significant ethical concerns.
    [Show full text]
  • Molecular Phylogenetic Analysis
    Assignment #2 Physics 498: Statistical Physics of Biological Complexity and Information Molecular Phylogenetic Analysis Date of Submission: 31th October, 2001 Rahul Roy Center for Biophysics and Computational Biology University of Illinois at Urbana-Champaign email: [email protected] Evolve by Borrowing ? Rahul Roy Center for Biophysics and Computational Biology University of Illinois at Urbana Champaign Introduction Molecular evolution over a period of four billion years has resulted in the development of present myriad of species from a hot soup of molecules. It has resulted in the evolution of complex multicellular organisms (Eukaryotes) along with single-celled organisms like prokaryotes at the same time. The question that has baffled scientists for long is: how did this incredible transition from a soup to this complex state take place? The discussion, that still continues, as how life started from the primordial soup is not the emphasis of the present debate, though Stanley Miller showed in, as back as 1950s, that organic chemicals can be synthesized from completely inorganic inputs. It has been proposed based on structural and functional complexity and fossil evidence that prokaryotes must have predated the eukaryotes by at least 1.0-2.0 billion years [Knoll, 1992]. This has led to the notion that eukaryotic cells have evolved from more simple prokaryotic organisms with which they share numerous common (or related) molecules [Margulis, 1970; Zillig, 1991]. Indeed, studies have shown that a number of eukaryotic organelles (namely mitochondria and chloroplasts) bear a close evolutionary relationship to specific groups of prokaryotes (namely α-proteobacteria and cyanobacteria, respectively) [Gray, 1992] So, how did the complex multicellular eukaryotes evolve from prokaryotes? To understand the origin of the eukaryotic cells a number of theories have been proposed.
    [Show full text]
  • BWPP Biological Weapons Reader
    BWPP Biological Weapons Reader Edited by Kathryn McLaughlin and Kathryn Nixdorff Geneva 2009 About BWPP The BioWeapons Prevention Project (BWPP) is a global civil society activity that aims to strengthen the norm against using disease as a weapon. It was initiated by a group of non- governmental organizations concerned at the failure of governments to act. The BWPP tracks governmental and other behaviour that is pertinent to compliance with international treaties and other agreements, especially those that outlaw hostile use of biotechnology. The project works to reduce the threat of bioweapons by monitoring and reporting throughout the world. BWPP supports and is supported by a global network of partners. For more information see: http://www.bwpp.org Table of Contents Preface .................................................................................................................................................ii Abbreviations .....................................................................................................................................iii Chapter 1. An Introduction to Biological Weapons ......................................................................1 Malcolm R. Dando and Kathryn Nixdorff Chapter 2. History of BTW Disarmament...................................................................................13 Marie Isabelle Chevrier Chapter 3. The Biological Weapons Convention: Content, Review Process and Efforts to Strengthen the Convention.........................................................................................19
    [Show full text]
  • Formation and Longevity of Chimeric and Duplicate Genes in Drosophila Melanogaster
    Copyright Ó 2009 by the Genetics Society of America DOI: 10.1534/genetics.108.091538 Formation and Longevity of Chimeric and Duplicate Genes in Drosophila melanogaster Rebekah L. Rogers,1 Trevor Bedford2 and Daniel L. Hartl Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138 Manuscript received May 15, 2008 Accepted for publication November 11, 2008 ABSTRACT Historically, duplicate genes have been regarded as a major source of novel genetic material. However, recent work suggests that chimeric genes formed through the fusion of pieces of different genes may also contribute to the evolution of novel functions. To compare the contribution of chimeric and duplicate genes to genome evolution, we measured their prevalence and persistence within Drosophila melanogaster. We find that 80.4 duplicates form per million years, but most are rapidly eliminated from the genome, leaving only 4.1% to be preserved by natural selection. Chimeras form at a comparatively modest rate of 11.4 per million years but follow a similar pattern of decay, with ultimately only 1.4% of chimeras preserved. We propose two mechanisms of chimeric gene formation, which rely entirely on local, DNA-based mutations to explain the structure and placement of the youngest chimeric genes observed. One involves imprecise excision of an unpaired duplication during large-loop mismatch repair, while the other invokes a process akin to replication slippage to form a chimeric gene in a single event. Our results paint a dynamic picture of both chimeras and duplicate genes within the genome and suggest that chimeric genes contribute substantially to genomic novelty.
    [Show full text]
  • Infectious Enveloped RNA Virus Antigenic Chimeras (Alphaviruses/Random Mutagenesis/Virus Vectors/Protein Engineering/Glycoproteins) STEVEN D
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 207-211, January 1992 Biochemistry Infectious enveloped RNA virus antigenic chimeras (alphaviruses/random mutagenesis/virus vectors/protein engineering/glycoproteins) STEVEN D. LONDON*, ALAN L. SCHMAUOHNt, JOEL M. DALRYMPLEt, AND CHARLES M. RICE*t *Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, MO 63110-1093; and tUnited States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011 Communicated by Edwin D. Kilbourne, October 7, 1991 (receivedfor review August 26, 1991) ABSTRACT Random insertion mutagenesis has been used would allow recovery of infectious chimeric viruses. The to construct infectious Sindbis virus structural protein chime- insertional mutagen was an oligonucleotide encoding an ras containing a neutralization epitope from a heterologous 11-amino acid neutralization epitope (4D4) derived from the virus, Rift Valley fever virus. Insertion sites, permissive for external G2 glycoprotein of Rift Valley fever virus (RVFV). recovery of chimeric viruses with growth properties similar to Both a polyclonal anti-peptide antiserum and a monoclonal the parental virus, were found in the virion E2 glycoprotein and antibody (mAb), mAb 4D4, that react with this epitope on the secreted E3 glycoprotein. For the E2 chimeras, the epitope RVFV virions and also with denatured and reduced RVFV was expressed on the virion surface and stimulated a partially G2 (13, 14) are available. Thus,
    [Show full text]
  • Recombinant VACV Fc Chimera (A.A
    Recombinant VACV Fc Chimera (a.a. 17-258, 18- 258, 100-330) [His] (DAG2641) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Product Overview Recombinant Viral CCI Fc Chimera antigen, was expressed in Spodoptera frugiperda, Sf 21 (baculovirus). N-terminus Viral CCI (Met17-Val258) & (Pro18-Val258) (Accession # , P19063), C- terminus Human IgG1 (Pro100-Lys330) and 6-His tag, Disulfide-linked homod Nature Recombinant Expression System Insect cells Species VACV Purity > 90%, by SDS-PAGE under reducing conditions and visualized by silver stain. Conjugate His Procedure None Format Lyophilized from a 0.2 μm filtered solution in PBS Preservative None Storage 2-8°C short term, -20°C long term BACKGROUND Introduction Vaccinia virus is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, and which encodes for approximately 250 genes. The dimensions of the virion are roughly 360 × 270 × 250 nm, with a mass of approximately 5-10 fg. Vaccinia virus is well known for its role as a vaccine (its namesake) that eradicated the smallpox disease, making it the first human disease to be successfully eradicated by science. This endeavour was carried out by the World Health Organization under the Smallpox Eradication Program. Post eradication of smallpox, scientists study Vaccinia virus to use as a tool for delivering genes into biological tissues (gene therapy and genetic engineering). Keywords Vaccinia virus vCCI protein, Human IgG1 protein; Orthopoxvirus; Vaccinia virus; 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved Chordopoxvirinae; VACV; VV 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 2 © Creative Diagnostics All Rights Reserved.
    [Show full text]
  • Chimeric Brains Consisted of Primatologists and Other Scientists, Ethicists, and Lawyers
    Chimeras Definition Ethical Issues Links Suggested Reading End Notes Definition: What are Chimeras? How and why are they produced? Chimeras are animals composed of cells that originate from two (or more) different species. In the research lab, chimeras are created by introducing cells from one species into the developing embryo or fetus of another. (The name chimera comes from Greek mythology and describes a creature with the head of a lion, the body of a goat, and the tail of a serpent). The first chimeras helped scientists understand questions about developmental biology. A sheep-goat chimera, created in 1984, had the head of a goat and the woolly coat of a sheep.i Chicken-quail chimeras have also been successfully developed.ii,iii Sheep-Goat Chimera Courtesy of Dr. Gary B. Anderson -University of California, Davis Now, researchers are developing human-animal chimeras to study disease processes, test new drugs, and develop organs for future transplant patients. The chimeras are produced by introducing human stem cells into developing animal embryos. The following are some of the major projects utilizing human- animal chimeras underway in the U.S and internationally: Sheep-to-humans organ transplant project: At the University of Nevada at Reno, researchers have added human stem cells to sheep fetuses to create chimeras that they hope will someday serve as a reliable source of organs for liver transplant patients. Some sheep now have livers with up to 80% human cells that produce the compounds normally made by human livers. (The cells are not all in distinct liver lobes but spread throughout the tissue.
    [Show full text]
  • A Computational Analysis to Construct a Potential Post-Exposure Therapy Against Pox Epidemic Using Mirnas in Silico
    orism terr & io B B i f o o d l e Hasan et al., J Bioterror Biodef 2016, 7:1 a f e n n r s u e DOI: 10.4172/2157-2526.1000140 o J Journal of Bioterrorism & Biodefense ISSN: 2157-2526 Research article Open Access A Computational Analysis to Construct a Potential Post-Exposure Therapy against Pox Epidemic Using miRNAs in Silico Mahmudul Hasan, Ewen McLean, and Omar Bagasra* South Carolina Centre for Biotechnology, Department of Biology, Claflin University, Orangeburg, SC 29115, USA Abstract Background: Smallpox was caused by Variola Virus (VARV) and even though it was eradicated in 1979, there exists the possibility of a zoonotic epidemic from Ortho-pox that may be pathogenic to humans. Moreover, VARV may still be used as a bioterrorist weapon. Monkey Pox Virus (MPXV), which is closely related to smallpox, is endemic to certain parts of Africa where sporadic outbreaks in humans are reported. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox caused by an Ortho Pox Virus (OPXV) related to MPXV, and cessation of routine smallpox vaccination with the live Vaccinia Virus (VACV), there is an increasing population of people susceptible to VARV and perhaps certain other zoonotic OPXV diseases. Were OPXV to be employed as a bioterrorist weapon, there is distinct possibility that it would be deployed as a chimera pox, with OPXV genes being combined with those of other poxviruses (e.g., MPXV, Tatera pox, etc). In such a case, the currently approved smallpox vaccine VACV, may be ineffective.
    [Show full text]
  • Long-Read DNA Metabarcoding of Ribosomal Rrna in the Analysis of Fungi from Aquatic Environments
    bioRxiv preprint doi: https://doi.org/10.1101/283127; this version posted March 15, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Long-read DNA metabarcoding of ribosomal rRNA in the analysis of fungi from aquatic environments Felix Heeger1,2†, Elizabeth C. Bourne1,2†, Christiane Baschien3, Andrey Yurkov3, Boyke Bunk3, Cathrin Spröer3, Jörg Overmann3, Camila J. Mazzoni2,4‡, Michael T. Monaghan1,2‡ 1Leibniz Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301, 12587 Berlin, Germany 2Berlin Center for Genomics in Biodiversity Research, Königin-Luise-Str. 6-8, 12489 Berlin, Germany 3Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7 B, 38124 Braunschweig, Germany 4Leibniz Institute of Zoo- and Wildlife Research (IZW), Alfred-Kowalke-Straße 17, 10315 Berlin, Germany † these authors contributed equally to this work ‡ these authors contributed equally to this work KEYWORDS: aquatic, biodiversity, CCS, chimera formation, eukaryotes, freshwater, fungi, isolates, long DNA barcodes, metabarcoding, mock community, Pacifc Biosciences, SMRT ABSTRACT DNA metabarcoding is now widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints have limited most studies to marker lengths of ca. 300-600 bp. Longer sequencing reads of several 5 thousand bp are now possible with third-generation sequencing. The increased marker lengths provide greater taxonomic resolution and enable the use of phylogenetic methods of classifcation, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most well- established bioinformatics tools for DNA metabarcoding were originally 10 designed for short reads and are therefore not suitable.
    [Show full text]
  • Chimeric 16S Rrna Sequence Formation and Detection in Sanger and 454-Pyrosequenced PCR Amplicons
    Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Resource Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons Brian J. Haas,1,9 Dirk Gevers,1 Ashlee M. Earl,1 Mike Feldgarden,1 Doyle V. Ward,1 Georgia Giannoukos,1 Dawn Ciulla,1 Diana Tabbaa,1 Sarah K. Highlander,2,3 Erica Sodergren,4 Barbara Methe´,5 Todd Z. DeSantis,6 The Human Microbiome Consortium, Joseph F. Petrosino,2,3 Rob Knight,7,8 and Bruce W. Birren1 1Genome Sequencing and Analysis Program, The Broad Institute, Cambridge, Massachusetts 02142, USA; 2Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA; 3Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA; 4The Genome Center, Washington University School of Medicine, St. Louis, Missouri 63108, USA; 5Human Genomic Medicine, J. Craig Venter Institute, Rockville, Maryland 20850, USA; 6Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA; 7Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA; 8Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309, USA Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) se- quences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short se- quences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets.
    [Show full text]
  • Detection of a New Resistance-Mediating Plasmid Chimera in a Blaoxa-48-Positive Klebsiella Pneumoniae Strain at a German University Hospital
    microorganisms Article Detection of a New Resistance-Mediating Plasmid Chimera in a blaOXA-48-Positive Klebsiella pneumoniae Strain at a German University Hospital Julian Schwanbeck 1,† , Wolfgang Bohne 1,†, Ufuk Hasdemir 2 , Uwe Groß 1, Yvonne Pfeifer 3, Boyke Bunk 4, Thomas Riedel 4,5, Cathrin Spröer 4, Jörg Overmann 4,5, Hagen Frickmann 6,7,‡ and Andreas E. Zautner 1,*,‡ 1 Institute for Medical Microbiology, University Medical Center Göttingen, 37075 Göttingen, Germany; [email protected] (J.S.); [email protected] (W.B.); [email protected] (U.G.) 2 Department of Medical Microbiology, School of Medicine, Marmara University, 34854 Istanbul, Turkey; [email protected] 3 Robert Koch Institute, FG13 Nosocomial Infections and Antibiotic Resistance, 38855 Wernigerode, Germany; [email protected] 4 Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, 38124 Braunschweig, Germany; [email protected] (B.B.); [email protected] (T.R.); [email protected] (C.S.); [email protected] (J.O.) 5 German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 38124 Braunschweig, Germany 6 Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, 20359 Hamburg, Germany; [email protected] Citation: Schwanbeck, J.; Bohne, W.; 7 Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Hasdemir, U.; Groß, U.; Pfeifer, Y.; 18057 Rostock, Germany Bunk, B.; Riedel, T.; Spröer, C.; * Correspondence: [email protected]; Tel.: +49-551-39-65927 Overmann, J.; Frickmann, H.; et al. † These authors contributed equally to this work. Detection of a New ‡ These authors contributed equally to this work. Resistance-Mediating Plasmid Chimera in a blaOXA-48-Positive Abstract: Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance Klebsiella pneumoniae Strain at a genes in Enterobacterales.
    [Show full text]
  • Human-Animal Chimeras
    Issue Brief June 2021 | No. IF21F01 Human-Animal Chimeras: Unethical and Unnecessary by Mary Szoch, M.Ed. Key Points n the United States, the ethical considerations regarding Human-animal chimeras are human-animal chimeras and human embryo research are creatures that are part human and part animal. Since 1979, intertwined. Understanding the regulations on human- there has been a restriction, Ianimal chimeras the United States ought to have requires a known as the 14-Day Rule, on developing this type of review of the United States’ policies on human embryo creature past 14 days gestation research, an overview of what human-animal chimeras are, an in vitro. analysis of the purported benefits of human-animal chimera In 2016, after scientists gained the ability to develop an research, and a synopsis of the ethical questions posed by embryo in vitro beyond nine creating these creatures. days, the National Institutes of Health revisited the 14-Day Rule, but under the pro-life Trump administration, the Rule The serious ethical concerns about the creation of human- was kept in place. animal chimeras and the lack of any beneficial scientific After a partnership with China advancements from chimera experiments make it clear that created the first human- primate chimera, the NIH, human-animal chimeras are unethical and unnecessary. under the Biden administration, opted to once again review the 14-Day Rule. Senators recently voted along party lines against banning the creation of human- animal chimeras through legislation. This report can be read online at frc.org/chimeras 801 G. St. NW Washington, D.C.
    [Show full text]