METHODS IN MOLECULAR BIOLOGY BIOLOGYTMTM Volume 269 VVacciniaaccinia VirusVirus andand PoxvirologyPoxvirology MethodsMethods andand ProtocolsProtocols Edited by Stuart N. Isaacs Vaccinia Virus and Poxvirology M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, SERIES EDITOR 291. Molecular Toxicology Protocols, edited by 270. Parasite Genomics Protocols, edited by Sara Phouthone Keohavong and Stephen G. Grant, E. Melville, 2004 2005 269. Vaccina Virus and Poxvirology: Methods 290. Basic Cell Culture, Third Edition, edited by and Protocols,edited by Stuart N. Isaacs, 2004 Cheryl D. Helgason and Cindy Miller, 2005 268. Public Health Microbiology: Methods and Protocols, edited by John F. T. Spencer and 289. Epidermal Cells, Methods and Applications, Alicia L. Ragout de Spencer, 2004 edited by Kursad Turksen, 2004 267. Recombinant Gene Expression: Reviews and 288. Oligonucleotide Synthesis, Methods and Appli- Protocols, Second Edition, edited by Paulina cations, edited by Piet Herdewijn, 2004 Balbas and Argelia Johnson, 2004 287. Epigenetics Protocols, edited by Trygve O. 266. Genomics, Proteomics, and Clinical Tollefsbol, 2004 Bacteriology: Methods and Reviews, edited 286. Transgenic Plants: Methods and Protocols, by Neil Woodford and Alan Johnson, 2004 edited by Leandro Peña, 2004 265. RNA Interference, Editing, and 285. Cell Cycle Control and Dysregulation Modification: Methods and Protocols, edited Protocols: Cyclins, Cyclin-Dependent Kinases, by Jonatha M. Gott, 2004 and Other Factors, edited by Antonio Giordano 264. Protein Arrays: Methods and Protocols, and Gaetano Romano, 2004 edited by Eric Fung, 2004 284. Signal Transduction Protocols, Second Edition, 263. Flow Cytometry, Second Edition, edited by edited by Robert C. Dickson and Michael D. Mendenhall, 2004 Teresa S. Hawley and Robert G. Hawley, 2004 283. Bioconjugation Protocols, edited by Christof 262. Genetic Recombination Protocols, edited by M. Niemeyer, 2004 Alan S. Waldman, 2004 282. Apoptosis Methods and Protocols, edited by 261. Protein–Protein Interactions: Methods and Hugh J. M. Brady, 2004 Applications, edited by Haian Fu, 2004 281. Checkpoint Controls and Cancer, Volume 260. Mobile Genetic Elements: Protocols and 2: Activation and Regulation Protocols, edited Genomic Applications, edited by Wolfgang J. by Axel H. Schönthal, 2004 Miller and Pierre Capy, 2004 280. Checkpoint Controls and Cancer, Volume 259. Receptor Signal Transduction Protocols, 1: Reviews and Model Systems, edited by Axel Second Edition, edited by Gary B. Willars H. Schönthal, 2004 and R. A. John Challiss, 2004 279. Nitric Oxide Protocols, Second Edition, ed- 258. Gene Expression Profiling: Methods and ited by Aviv Hassid, 2004 Protocols, edited by Richard A. Shimkets, 2004 278. Protein NMR Techniques, Second Edition, edited by A. Kristina Downing, 2004 257. mRNA Processing and Metabolism: Methods and Protocols, edited by Daniel R. Schoenberg, 277. Trinucleotide Repeat Protocols, edited by 2004 Yoshinori Kohwi, 2004 256. Bacterial Artifical Chromosomes, Volume 276. Capillary Electrophoresis of Proteins and 2: Functional Studies, edited by Shaying Zhao Peptides, edited by Mark A. Strege and and Marvin Stodolsky, 2004 Avinash L. Lagu, 2004 255. Bacterial Artifical Chromosomes, Volume 275. Chemoinformatics, edited by Jürgen Bajorath, 1: Library Construction, Physical Mapping, 2004 and Sequencing, edited by Shaying Zhao and 274. Photosynthesis Research Protocols, edited Marvin Stodolsky, 2004 by Robert Carpentier, 2004 254. Germ Cell Protocols, Volume 2: Molecular 273. Platelets and Megakaryocytes, Volume 2: Embryo Analysis, Live Imaging, Transgenesis, Perspectives and Techniques, edited by and Cloning, edited by Heide Schatten, 2004 Jonathan M. Gibbins and Martyn P. Mahaut- 253. Germ Cell Protocols, Volume 1: Sperm and Smith, 2004 Oocyte Analysis, edited by Heide Schatten, 2004 272. Platelets and Megakaryocytes, Volume 1: 252. Ribozymes and siRNA Protocols, Second Functional Assays, edited by Jonathan M. Edition, edited by Mouldy Sioud, 2004 Gibbins and Martyn P. Mahaut-Smith, 2004 251. HPLC of Peptides and Proteins: Methods and 271. B Cell Protocols, edited by Hua Gu and Klaus Protocols, edited by Marie-Isabel Aguilar, 2004 Rajewsky, 2004 M E T H O D S I N M O L E C U L A R B I O L O G Y™ Vaccinia Virus and Poxvirology Methods and Protocols Edited by Stuart N. Isaacs Division of Infectious Diseases, University of Pennsylvania Philadelphia, PA Foreword by Bernard Moss National Institutes of Health, NIAID Laboratory of Viral Diseases, Bethesda, MD Humana Press Totowa, New Jersey © 2004 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permis- sion from the Publisher. Methods in Molecular Biology™ is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ' ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Patricia F. Cleary. Cover illustrations: Background: Figure 4, Chapter 5, X.-D. Yao and D. H. Evans. Foreground top: Smallpox leasions on skin and trunk. Photo taken in Bangladesh. Photo source: CDC Public Health Image Library (http://phil.cdc.gov). Foreground bottom left: Figure 3, Chaper 2, M. M. Lorenzo, I. Galindo, and R. Blasco. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Vaccinia virus and poxvirology : methods and protocols / edited by Stuart N. Isaacs ; foreword by Bernard Moss. p. ; cm. — (Methods in molecular biology ; 269) Includes bibliographical references and index. ISBN 1-58829-229-0 (alk. paper) eISBN 1-59259-789-0 1. Vaccinia—Laboratory manuals. 2. Recombinant poxviruses—Laboratory manuals. [DNLM: 1. Vaccinia virus—genetics. 2. DNA Replication. 3. Molecular Biology—methods. 4. Poxviridae—genetics. 5. Vaccinia virus—immunology. 6. Virus Replication. QW 165.5.P6 V116 2004] I. Isaacs, Stuart Neal. II. Series: Methods in molecular biology Clifton, N.J.) ; v. 269. QR412.V33 2004 579.2’432—dc22 2003023492 Foreword The Right Book at the Right Time The poxviruses comprise a family of complex DNA viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. Of the eight recognized gen- era of vertebrate poxviruses, those belonging to the orthopoxvirus genus have been most intensively studied. This group includes variola virus, the agent of smallpox, as well as cowpox virus and vaccinia virus. Jenner’s original small- pox vaccine, described in 1798, consisted of live cowpox virus, but vaccinia virus later replaced it (1). There has been speculation as to the origin of vac- cinia virus; the most likely idea is that it is a separate species, possibly origi- nally isolated from a horse, and is now extinct or rare in nature (2). Recent genome sequencing studies confirm the distinctness of variola virus, cowpox virus, and vaccinia virus and also their very close genetic relationship, which accounts for the cross protection of smallpox vaccines. The novelty of the smallpox vaccine can be readily appreciated by the time it took, about 80 years, before the next live vaccine against rabies was developed, and another 50 years for the yellow fever vaccine. Moreover, the eradication of smallpox in 1977 stands as a unique medical achievement. Because of its historical role, small- pox vaccination contributed greatly to present concepts of infectious disease, immunity, and pathogenesis. Less well known, however, are the many other “firsts” for vaccinia virus. Vaccinia virus was the first animal virus seen micro- scopically, grown in tissue culture, accurately titrated, physically purified, chemically analyzed, shown to regulate gene expression, and found to encode transcriptase and replicase activities and modulators of host defense (3). Through the early 1980s, a relatively small number of laboratories worked with vaccinia virus. This number increased dramatically following the demon- stration of vaccinia virus as a gene expression vector (4,5) and the wide dis- semination of convenient transfer plasmids for the generation of recombinant vaccinia viruses by homologous recombination (6). These recombinant viruses were used for a myriad of purposes