Table Of ContentMETHODS IN VIROLOGY
Advisory Board
F. C. BAWDEN ROY MARKHAM
ROTHAMSTED EXPERIMENTAL STATION JOHN INNES INSTITUTE
HARPENDEN, HERTS, ENGLAND NORWICH, NORFOLK, ENGLAND
SVEN GARD
K. F. MEYER
INSTITUTIONEN FUR VIRUSFORSKNING
SAN FRANCISCO MEDICAL CENTER
KAROLINSKA INSTITUTET
GEORGE WILLIAMS HOOPER FOUNDATION
STOCKHOLM, SWEDEN
SAN FRANCISCO, CALIFORNIA
GEORGE K. HIRST
GEORGE E. PALADE
PUBLIC HEALTH RESEARCH INSTITUTE
OF THE CITY OF NEW YORK, INC. THE ROCKEFELLER UNIVERSITY
NEW YORK, NEW YORK NEW YORK, NEW YORK
S. E. LURIA
C. VAGO
DEPARTMENT OF BIOLOGY
INSTITUTE OF CYTOPATHOLOGY
MASSACHUSETTS INSTITUTE
SAINT CHRISTOL, GARD, FRANCE
OF TECHNOLOGY
CAMBRIDGE, MASSACHUSETTS ROBLEY C. WILLIAMS
ANDRE LWOFF
VIRUS LABORATORY
INSTITUTE PASTEUR UNIVERSITY OF CALIFORNIA
PARIS, FRANCE BERKELEY, CALIFORNIA
METHODS IN VIROLOGY
EDITED BY
KARL MARAMOROSCH
BOYCE THOMPSON INSTITUTE FOR PLANT RESEARCH
YONKERS, NEW YORK
AND
HILARY KOPROWSKI
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PENNSYLVANIA
Volume V
ACADEMIC PRESS New York and London 1971
COPYRIGHT © 1971, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED
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PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
MILTON ADESNIK, Department of Biological Sciences, Columbia University,
New York, New York (125)
ELLEN M. BALL, Department of Plant Pathology, University of Nebraska
Lincoln, Nebraska (445).
ALMEN L. BARRON, Department of Microbiology, State University of New
York at Buffalo, Buffalo, New York (347).
HEINZ BAUER, Department of Bacteriology and Immunology, University
of Western Ontario, London, Ontario, Canada (481).
KJELL B0VRE, Bakteriologiske Institutt, Rikshospitalet, Oslo, Norway (271).
SYDNEY S. BREESE, JR., U. S. Department of Agriculture, Plum Island
Animal Disease Laboratory, Greenport, Long Island, New York
(399).
MAURICE GREEN, Institute for Molecular Virology, Saint Louis University
School of Medicine, Saint Louis, Missouri (247).
KONRAD C. Hsu, Department of Microbiology, Columbia University,
New York, New York (399).
CLYDE A. HUTCHISON, III, Department of Bacteriology and Immunology,
School of Medicine, University of North Carolina, Chapel Hill,
North Carolina (79).
EDOUARD KURSTAK, Laboratoire de Virologie Comparee, Department de
Microbiologic et Immunologic, Faculte de Medecine, Universite de
Montreal, Quebec, Canada (423).
HOMER A. LOZERON, McArdle Laboratory, University of Wisconsin,
Madison, Wisconsin (271).
JACOB V. MAIZEL, JR., Department of Cell Biology, The Albert Einstein
College of Medicine, New York, New York (179).
BENJAMIN MANDEL, Department of Virology, The Public Health Research
Institute of the City of New York, New York (375).
EDWARD L. MEDZON, Department of Bacteriology and Immunology,
University of Western Ontario, London, Ontario, Canada (481).
JOSEPH S. PAGANO, Department of Medicine and Department of Bacteri
ology and Immunology, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina (79).
xi
xii LIST OF CONTRIBUTORS
A. POLSON, M. R. C. & U. C. T. Virus Research Unit, Medical School,
Observatory, Cape Town, South Africa (33).
HESCHEL J. RASKAS, Institute for Molecular Virology, Saint Louis Univer
sity, School of Medicine, Saint Louis, Missouri (247).
J. S. SALSER, Division of Biological Chemistry, Sloan-Kettering Institute
for Cancer Research, Sloan-Kettering Division Graduate School of
Medical Sciences, Cornell University, New York (293).
WACLAW SZYBALSKI, McArdle Laboratory, University of Wisconsin,
Madison, Wisconsin (271).
J. F. WATKINS, Sir William Dunn School of Pathology, Oxford, England (1).
C. E. YARWOOD, Department of Plant Pathology, University of California,
Berkeley, California (451).
Preface
In a rapidly developing field of scientific endeavor such as virus research,
technical approaches, which are of paramount importance for progress in
the field, are not static but undergo constant change. Although most of
the methods described in the first four volumes of "Methods in Virology"
still remain popular with scientists working in the field, many of the tech
niques have been simplified and improved since the publication of these
volumes. At the same time, new approaches to the solution of many prob
lems have called for the development of new techniques, hence, the need
for a new volume in this treatise. Since neither the editors nor the publisher
are endowed with prophetic properties, it was difficult to select those
methods which would find their application not only today but also in
the near or even more distant future. The editors, however, in their choice
of techniques, hope they will prove useful in the virus research of today
and of tomorrow.
As in the first four volumes, the chapters in Volume V are self-contained,
and some overlapping of information could not be avoided. Each chapter
is followed by extensive literature citation so that the reader will be able to
find all the pertinent references to a given method without difficulty. Certain
chapters are complete treatises on a given subject, while others provide a
description of techniques more limited in scope, therefore requiring a more
elaborate interpretation of the results.
The editors wish to thank their Board of Advisors, particularly S. E.
Luria and Robley C. Williams, for their invaluable assistance in the prep
aration of this volume. Last, but not least, thanks are due Academic Press
for their part in the production aspects of this volume.
KARL MARAMOROSCH
HILARY KOPROWSKI
xiii
Contents of Other Volumes
Volume I
Natural Ecology Methods for Experimenting with Mite
Harald Norlin Johnson Transmission of Plant Viruses
Virus Hosts and Genetic Studies J. T. Slykhuis
A. G. Dickinson and J. M. K. Mackay Fungus Transmission of Plant Viruses
Methods for the Study of Mosquitoes as D. S. Teakle
Virus Hosts and Vectors Plant Viruses: Transmission by Dodder
Roy W. Chamberlain and W. Daniel Sudia C. W. Bennett
Methods of Studying Ticks and Mites as Graft Transmission of Plant Viruses
Virus Hosts and Vectors L. Bos
Cornelius B. Philip Insect Pathogenic Viruses
Methods of Studying Plants as Virus Hosts Kenneth M. Smith
L. Bos Bacteriophage Techniques
Laboratory Methods of Virus Transmission A. Eisenstark
in Multicellular Organisms Animal Tissue Culture
D. Blaskovic and B. Styk J. S. Porterfield
Mechanical Transmission of Plant Viruses Plant Tissue Culture
C. E. Yarwood and R. W. Fulton B. Kassanis
Plant Virus Transmission by Insects Invertebrate Tissue Cultures
K. G. Swenson C. Vago
Nematode Transmission AUTHOR INDEX—SUBJECT INDEX
D. J. Raski and W. B. Hewitt
Volume II
The Ultracentrifuge Diffusion
Roy Markham Roy Markham
Equilibrium Ultracentrifugation Two-Phase Separation of Viruses
H. M. Mazzone Per-Ake Albertsson
Density-Gradient Centrifugation Purification of Virus by Adsorption on Cells
Myron K. Brakke and Elution
Miscellaneous Problems in Virus Purification Felix E. Wassermann
Myron K. Brakke Molecular Sieve Methods
New Centrifugal Methods for Virus Isolation G. K. Ackers and R. L. Steere
N. G. Anderson and G. B. Cline Filtration Techniques
Chromatography and Membrane Separation Vernon P. Perry and Monroe M. Vincent
Lennart Philipson Electrophoresis of Viruses
Water-Organic Solvent Phase Systems A. Poison and B. Russell
Lennart Philipson Labeling of Viruses with Isotopes
Virus Concentration by Ultrafiltration Claudia Henry
Karl Strohmaier
xv
xvi CONTENTS OF OTHER VOLUMES
Volume II {Continued)
Separation of Viruses into Components Assay of Infectivity of Nucleic Acids
R. K. Ralph and P. L. Bergquist S. Sarkar
Methods of Degrading Nucleic Acids and AUTHOR INDEX—SUBJECT INDEX
Separating the Components
T. H. Lin and R. F. Maes
Volume III
Analysis of Protein Constituents of Viruses Methods for Selecting RNA Bacteriophage
H. Fraenkel-Conrat and R. R. Rueckert Mamoru Watanabe and J. T. August
Analysis of Lipid Components of Viruses Structural Studies of Viruses
David Kritchevsky and Irwin L. Shapiro J. T. Finch and K. C. Holmes
RNA Virus RNA Polymerase: Detection, Microscopic Techniques
Purification, and Properties Rex S. Spendlove
J. T. August and Lillian Eoyang Electron Microscopy of Isolated Virus Par
Immunological Techniques for Animal ticles and Their Components
Viruses Robert W. Home
Jordi Casals The Application of Thin Sectioning
Serological Techniques for Plant Viruses C. Morgan and H. M. Rose
R. E. F. Matthews Autoradiographic Methods for Electron
The Plaque Assay of Animal Viruses Microscopy
Peter D. Cooper Nicole Granboulan
Transformation Assays AUTHOR INDEX—SUBJECT INDEX
M. G. P. Stoker and I. A. Macpherson
Volume IV
Techniques for the Study of Interferons in Methods for the Study of Defective Viruses
Animal Virus-Cell Systems Hidesaburo Hanfusa
Robert R. Wagner, Allan H. Levy, and Cell Cultures and Pure Animal Virus in
Thomas J. Smith Quantity
Methods for the Study of Viral Inhibitors Howard L. Bachrach and
Felix E. Wassermann Sydney S. Breese, Jr.
Methods of Inactivation by Ultraviolet Methods in Human Virus Vaccine Prepara
Radiation tion
A. Kleczkowski Louis Potash
Inactivation of Viruses by Ionizing Radiation Methods for Containment of Animal Path
and by Heat ogens at the Plum Island Animal
William Ginoza Disease Laboratory
Methods for Testing Antiviral Agents Jerry J. Callis and George E. Cottral
Frantisek Link Methods of Storage and Preservation of
Techniques for Studying Defective Animal Viruses
Bacteriophages Thomas G. Ward
Allan M. Campbell
CONTENTS OF OTHER VOLUMES xvii
Volume IV (Continued)
Methods of Preservation and Storage of Methods for the Study of Colicine and
Plant Viruses Colicinogeny
Harold H. Mckinney and Gustave Silber Haruo Ozeki
The Optical Diffractometer Methods of Virus Classification
Roy Markham C. J. Andrewes
Contamination of Cell Cultures by Myco Experimental Design and Statistical Methods
plasma (PPLO) of Assay
Arthur Brown and Julius E. Officer A. Kleczkowski
AUTHOR INDEX—SUBJECT INDEX
1 Fusion of Cells for Virus Studies and
Production of Cell Hybrids
J. F. Watkins
I. Introduction 1
II. Methods 5
A. Preparation and inactivation of Sendai Virus 5
B. Cell Fusion with Inactivated Sendai Virus 8
C. Assessment of Fusion 11
III. Cell Fusion in Virology 19
A. Detection of Virus in Transformed Cells 19
B. Infection of Insusceptible Cells Using Cell Fusion 21
C. Cell Fusion and Studies on Interferon 21
IV. Selection of Hybrid Cell Lines 23
A. Physical Methods 23
B. Biochemical Methods 24
C. Other Possible Methods of Hybrid Selection 29
V. Uses of Heterokaryons and Hybrid Cell Lines 30
References 31
/. Introduction
Vertebrate cells can be fused in two ways, by "spontaneous" fusion, or
treatment with certain viruses. The former method was first described by
Barski et al. (1960), and requires the mixed cultivation of two continuous
lines of cells. Spontaneous mating events may occur with a low or com
paratively high frequency. For example, in mixed cultures of hamster mel
3
anoma cells and mouse cells the "mating rate" was between 10~ and
4
10" (Davidson et al., 1966). The latter method is based on the observa
tions of Okada (1962), who found that Ehrlich ascites tumor cells in sus
pension could be fused within an hour by mixing them with a myxovirus,
HVJ, and incubating at 37°C. Fusion also occurred when the virus had
l
