Jrtited States
flvironrnental Protection
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
EPA-600'1-80-026
May 1980
Research and Development
Investigation of the
Persistence and
Replication of
Nuclear Polyhedrosis
Viruses in
Vertebrate and
Insect Cell
Cultures by the
Use of Hybridization
Techniques
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series, These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series, This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-80-026
May 1980
INVESTIGATION OF THE PERSISTENCE AND REPLICATION
OF NUCLEAR POLYHEDROSIS VIRUSES IN VERTEBRATE AND INSECT
CELL CULTURES BY THE USE OF HYBRIDIZATION TECHNIQUES
by
William Meinke, D. A. Goldstein, Cynthia Alvidrez, and John Spizizen
Scripps Clinic and Research Foundation
10666 N. Torrey Pines Road
La Jolla, California 92037
and
C. B. William and H. R. Lukens
IRT Corporation
7650 Convoy Court
San Diego, California 92138
Contract No. 68-02-2209
Project Officer
C. Y. Kawanishi
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and Approved for publication. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the
establishment of sound regulatory policy. These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology, epidemio-
logy, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis, and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory develops and revises air quality criteria
documents on pollutants for which national ambient air quality standards
exist or are proposed, provides the data for registration of new pesticides
or proposed suspension of those already in use, conducts research on hazardous
and toxic materials, and is preparing the health basis for non-ionizing
radiation standards. Direct support to the regulatory function of the Agency
is provided in the form of expert testimony and preparation of affidavits as
well as expert advice to the Administrator to assure the adequacy of health
care and surveillance of persons having suffered imminent and substantial
endangerment of their health.
The majority of the registered pesticides are chemical agents. A few,
however, are biological in nature because the active ingredients are micro-
bial. Of these micro-organisms, viruses are perhaps the most unique in
structure, biology, and the intimacy of their parasitic relationship with
their hosts. This report considers whether potential biohazards to human
health and other biological components of the environment exist when instect
viruses are used as pesticides.
F. Gordon Hueter, Ph.D.
Director
Health Effects Research Laboratory
iii
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ABSTRACT
The goal of this research is to determine whether a nuclear polyhedrosis virus
(NPV) can interact with a variety of mammalian and insect cells in vivo, producing
either apparent or inapparent infection. The use of DNA-DNA hybridization techniques
to detect the presence of viral genomes in cells will provide a sensitive method of
monitoring viral persistence or replication, even in the absence of cytopathic effects or
the production of infectious virus particles. It is proposed that the DNA-DNA
hybridization technique be developed as a sensitive assay for the replication or
persistence of NPV in vitro, allowing a more effective evaluation of the safety of these
viruses as biological control agents than has been possible employing previously
established methods.
IV
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CONTENTS
Foreword lil
Abstract iv
Figures vi
1. Introduction 1
2. Background 2
3. Infectious Unit ^
Characterization of the tissue culutre infectious unit from
Trichoplusia ni (T. ni) cells infected with A. californica NPV ... 4
4. Materials and Methods 5
Cells 5
Virus 5
Tritium labeling of viral DNA in vivo 5
Purification of H-labeled virions 6
Infection of monkey kidney cells (CV-1) 7
Detection of PIB protein by indirect immunofluorescent
antibody staining 7
Electron microscopy 7
5. Results 9
Defining the infectious unit 9
Infection of monkey cells with A. californica NPV 19
6. To Determine Whether Free, Non-Occluded Autographica Californica
Nuclear Polyhedrosis Virus can Adsorb and Penetrate Mammalian
Cells in vitro 26
Preparation of H-labeled free non-occluded A. californica virions
in T. ni cells in vitro (used for infection of monkey cells) . .... 26
Infection of monkey kidney cell cultures with H-labeled A. cal.
non-occluded virions 28
Persistence of A. californica DNA in infected CV-1 monkey cells . 31
Preparation of in vitro labeled A. cal. NPV DNA 33
In vitro labeling of A. cal. NPV DNA employing E. coli DNA
polymerase 35
7. The Use of DNA-DNA Hybridization for Detection of the Persistence
of Viral Genomes 38
Isolation of viral DNA 38
Preparation of in vitro labeled viral DNA 38
Inspection of cell lines 42
Isolation of cellular DNA 42
DNA reassociation 42
8. Summary 45
9. References 46
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FIGURES
Number
1 Purification of H-labeled A. californica virus infected T. ni cells
48 hours post-infection 6
2 Incorporation of H-thymidine into non-occluded A. calif ornica NPV . . 10
3 Velocity sedimentation of virions in sucrose gradients 12
4 Velocity sedimentation analysis of virions treated with DNase and
detergents 13
5 Particles from sucrose gradients (rod-shaped "bags") with an average
length of about 317 nm and a diameter between about 40 and 80 nm
with PTA staining 15
6 Occluded dark-staining virus rods in sections of polyhedra which are
about 240 to 270 nm 16
7 Virus observed by uranyl acetate staining show "bags" which appear to
be "exploding" at both ends 17
8 Virus observed by uranyl acetate staining show "bags" which appear to
be "exploding" at both ends 18
9 Alkaline degradation of H-thymidine labeled A. calif ornica polyhedral
inclusion bodies 19
10 Morphological characteristics typical of polyhedra 21
11 Morphological characteristics typical of polyhedra 22
12 Bizarre morphology of monkey cells maintained at 28°C 23
13 Fluorescence in nuclei of infected T. ni cells 24
14 Control for T. ni cell fluorescence experiment 24
15 Velocity sedimentation of A. cal. virions in a sucrose gradient 27
16 Purification of H-labeled A. cal. non-occluded NPV 28
VI
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FIGURES (Continued)
Number Page
17 Uptake of radioactive A. cal. virions by CV-1 monkey cells and T. ni
insect cells ......................... 29
18 Sedimentation of an SDS lysate of CV-1 monkey cells six hours
following infection by H-labeled A. cal. virions ......... 30
19 Sedimentation of A. cal. NPV DNA extracted by alkali and SDS from
purified PIB ......................... 35
20 CsCl-dye equilibrium centrifugation of A. cal. viral DNA ....... 39
21 In vitro labeling of viral DNA ................... 40
22 Alkaline sucrose gradient centrifugation of in vitro labeled A. cal. DNA . 41
23 Schematic of extraction of cellular DNA .............. 43
24 Hybridization of in vitro labeled A. cal. DNA ............ 44
Vll
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SECTION 1
INTRODUCTION
This is a combined final report prepared under Contract 68-02-2209 by IRT
Corporation for the Environmental Protection Agency. The first part of the work was
previously published as IRT report INTEL-RT 8135-001, March 1977 and the second part
was published as IRT 8135-010 in January 1978.
This work was carried out in the Department of Microbiology, Scripps Clinic and
Research Foundation, and the authors are indebted to Dr. John Spizizen, Chairman, for
his support and encouragement.
Much of the experimental work reported was carried out by Robert Mandel with
considerable care and skill, and his contribution is hereby acknowledged.
The authors are indebted to Dr. Vail of the USDA, Phoenix, Arizona, and
Dr. Timothy Kurtti for provision of materials.
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SECTION 2
BACKGROUND
The rapid rise in world population and the subsequent demand for increased food
production has given great impetus to the development of safe and effective pesticides.
At the same time, the prolonged use of many chemical pesticides since World War II has
resulted in the development of insect populations resistant to the effects of many of
these pesticides which can harm nontarget organisms and result in the accumulation of
toxic chemical residues in the environment.
These problems have focused attention on the possibility of using arthropod
viruses as insect pesticides (Ref 2), especially the nuclear polyhedrosis (NPV) and
granulosis (GV) viruses. A report from the World Health Organization (WHO) lists more
than 400 viruses which are pathogens for insects and mites (Ref 3). These viruses are
extremely virulent for their insect hosts, but appear to have a narrow host range,
generally limited to a single genus or species (Ref 2). Viral pesticides thus seem to
offer the promise of control of defined insect pests without damaging other populations
or leaving toxic residues.
Before being licensed for widespread use as a pesticide, an insect virus must be
evaluated for any possible harmful effects to insects other than the target species,
other invertebrates, vertebrates (including man), and plants. Indirect evidence accumu-
lated during prolonged exposure of workers to NPV during commercial production of the
virus, field application, or ingestion in food indicate that NPV induced no clinical
symptoms or immunological response in exposed individuals (Ref 4). Numerous other in
vivo studies have indicated that arthropod NPV cause no deleterious effects on
bacteria, plants, invertebrates other than insects, and vertebrates, including numerous
fish, avian, and mammalian species (Refs 4-7). Inocula ranged from ten to 1,000 times
the normal field exposure of the virus, and the test species were observed for factors
including virally induced immunological response, toxicity, pathogenicity, teratogen-
icity, and carcinogenicity.
The in vitro specificity of NPV has also been investigated. The Autographa
californicaTA. cal.) NPV has been shown to be able to cross species lines and replicate
in cells from several different genera of Lepidoptera (Refs 8,9). Attempts to infect
vertebrate cell cultures with NPV have been unsuccessful with one exception. Himeno
et al. (Ref 10) reported the formation of polyhedral inclusion bodies (PIB) after
inoculation of FL (human amnion) cells with viral DNA from Bombyx mori NPV. Ignoffo
and Rafajko (Ref 11) could demonstrate no replication or cytopathic effects (CPE) of
the NPV of Heiiothis zea in four primate cell lines. A later study by Mclntosh and
Maramorosch (Ref 12) reported no deleterious effects of H. zea NPV infection in five
human cell lines inoculated with either PIB or virions released from PIB by mild alkali
treatment. Three of these cell lines did retain infectious NPV for up to four weeks
after inoculation.
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These in vitro studies were designed to observe productive NPV infection by
detecting the formation of infectious progeny virus or cytopathic effects such as ceil
rounding and PIB formation. A latent viral infection, or one proceeding without
demonstrable CPE, would not be apparent.
Ignoffo et ah (Ref 13) have demonstrated that HL zea NPV can be passed through
an established line of H^ zea cells without producing free virions or characteristic
inclusion bodies. Cell cultures were inoculated with viral DNA and carried through
seven consecutive cell passages. After seven cell passages, infectivity, measured by
injection of cotton boll worm larvae with culture extracts and subsequent production of
o
PIB and larval death, increased 10 times over that present in the original inoculum.
Inclusion bodies or virions were not detectable by electron microscopy in any of the
passages.
Two strains of Trichoplusia ni (T. ni) NPV have been isolated by plaque
morphology (Ref 14). One strain forming 30 or more polyhedra per nucleus was
designated MP (many polyhedra) while another producing less than ten polyhedra per
nucleus were classified as FP (few polyhedra). Serial passage of MP virus through tissue
culture cells led to the production of virus with the FP phenotype. The FP viral
inclusions were not lethal to larvae but displayed superior growth potential in vitro. FP
virions were released from in vitro cells more quickly and rose to higher titers than the
MP viral strain. Serial in vitro passage of this virus seemed to enhance its ability to
replicate in vitro, while decreasing the production of PIB and making the detection of
CPE more difficult.
These results suggest that techniques capable of determining the fate of the viral
genome in infected cell cultures are needed in order to accurately determine the host
range of NPV. The finding that viral genes are readily degraded in infected cell
cultures would support the belief that nuclear polyhedrosis viruses pose no serious
threat to vertebrates, including man and domestic animals. However, if viral genes
persist in infected cells, the possibility that certain insect viruses may be potentially
hazardous would have to be considered.
The fate of viral gene sequences in infected cell cultures can be followed by the
use of extremely sensitive DNA-DNA hybridization techniques (Refs 15,16). The
sensitivity of DNA-DNA hybridization is great enough to allow the detection of less
than one viral genome equivalent per 5-10 cells. In this procedure purified viral DNA is
labeled with radioactive nucleotides in vitro, denatured, and allowed to reassociate in
the presence of a vast excess of unlabeled DNA extracted from infected cells. The
presence of gene sequences homologous to viral genes in cellular DNA will increase the
rate of reassociation of the radioactively labeled viral DNA probe. The number of viral
genome equivalents present in the DNA from infected cells can be estimated from the
change in the reassociation rate (Ref 17).
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SECTION 3
INFECTIOUS UNIT
CHARACTERIZATION OF THE TISSUE CULTURE INFECTIOUS UNIT FROM
Trichoplusia ni (T. ni) CELLS INFECTED WITH A. calif ornica NPV
While nuclear polyhedrosis viruses have been propagated successfully in vitro in
insect tissue culture cells, unequivocal identification of the infectious unit has not been
achieved. The original inocula are usually derived from the hemolymph of infected
larvae. Once the infection is established, the virus is subsequently propagated by
infecting cultures with the overlay media from previously infected cultures. Although
it has been generally conceded that polyhedra inclusion bodies (PIB) obtained from
tissue culture cells have a low (or no) specific infectivity, it is not known whether the
actual infectious unit in the media is enveloped virions, naked virus rods, sub-virion
complexes, or naked DNA.
Since our ultimate goal is to detect infection of mammalian cells with insect
viruses, it is essential that the infectious entity be defined. To this end, a number of
experiments have been performed to isolate and characterize the infectious material
from overlay media, as well as from infected cells. The basic approach has been to tag
the virus by growing infected cells in the presence of radioactive precursors to DNA,
and to fractionate and characterize specific sub-cellular and extra-cellular fractions.
Various fractions were characterized by: (1) velocity sedimentation rate; (2) bouyant
density of fixed and unfixed material in CsCj^; (3) sensitivity to various enzymes and
detergents; (4) electron microscopy; and (5) infectivity in T. ni cells in tissue culture.
Growth media was removed from CV-1 cells, and they were covered with overlay media
taken from infected T. ni cells which contained the infectious entity. In different
cultures, virus was permitted to absorb for one hour at one of two temperatures, either
28 C or 37 C. The reason for infecting at 28°C is that this is the optimum growth
temperature for virus. The rationale for infecting at 37°C was that this is the optimum
growth temperature for the monkey cells. Cells were then overlayed with fresh monkey
cell growth media and the cultures monitored for signs of infection. These included
examination by immunofluorescence for the presence of virus-specific proteins and
observation for cytopathic effect by phase and electron microscopy. Attempts to
follow the uptake of radioactive virus were unsuccessful in this initial series of
experiments, since it was not possible to achieve a high enough incorporation of
radioactivity into virions to make this practical. This approach is now being repeated in
experiments in progress.
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SECTION 4
MATERIALS AND METHODS
CELLS
Monkey kidney cells (CV-1) were obtained from the American Type Tissue Culture
Association, and were grown as monolayer cultures in plastic petri dishes (100 mm).
Growth media was Dulbecco's Modified MEM (Gibco), supplemented with 10 percent
fetal bovine serum (Gibco) and incubated at 37°C in a 5 percent CO2 atmosphere. Cells
were routinely subcultured by trypsinization, and replated at a 1:3 or 1:* dilution every
four to five days.
Insect cells T. ni (TN-368), were supplied by Dr. W. Fred Hink. Cells were grown
in 25 cm Falcon plastic screw cap flasks in Hink's modification of Grace's Insect T.C.
medium, the medium designated TNM-FH. The antibiotics, penicillin (Lilly) 50,000
units/100 m^ and 10 mg/100 mJ! streptomycin SCX (Lilly) were added immediately
before use. Cells were grown at 28°C and routinely subcultured at a 1:5 dilution every
three days by removing the spent growth media, resuspending the cells (shaken off the
flask by violent agitation) into fresh growth media, and dispensing the cells into new
flasks.
VIRUS
The original A. californica nuclear polyhedrosis virus (NPV) was provided by
Dr. W. Fred Hink and consisted of growth media obtained from infected T. ni cells.
Subsequent inocula were prepared by harvesting growth media from infected cells and
removing high molecular weight material by centrifugation at 5,000 x g. This superna-
tant fluid containing the infectious unit was stored at 4°C or -70°C.
TRITIUM LABELING OF VIRAL DNA in vivo
Viral DNA was labled by the following procedure. T. ni cells were infected in the
logarithmic growth phase (70 to 80 percent confluent) by removing the media and
inoculating with 0.5 mj? of media containing infectious virus. This inoculum was a 1:10
dilution of a typical "growth media" inoculum described above. After one hour at 28°C
(standard infection procedure), virus was removed and fresh media added containing
•7 O
either (1) 10 \iCilrc\St H-deoxycytidine (110 mCi/mmole); (2) 10 f*Ci/W H-thymine
(15 Ci/mmole); (3) 10 nCi/mjC, H-deoxycytosine-5'-monophosphate (25.3 Ci/mmole); or
Ct) 20 /LtCi/m^ H-thymidine (52.6 Ci/mmole) (New England Nuclear). Cells were
maintained at 28°C, and harvested 48 hours after infection.
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PURIFICATION OF ^H-LABELED VIRIONS
Non-Occluded "Free" Virions
Growth media containing tritium-labeled precursors as described above
(e.g., either thymine, thymidine, deoxycytidine, or deoxycytidine monophosphate) were
processed by centrifugation at low speed (700 x g, 10 minutes, 4 C) to remove cell
debris, and then centrifuged at high speed (75,000 x g, 60 minutes, 23 C) to pellet the
"free" (as opposed to cell-associated) non-occluded virus. The pellet was resuspended in
a small volume of sucrose gradient buffer (SGB) (0.05 M Tris, pH 8.0, 0.3 M NaC?)
layered onto a linear 5.0 mjt, 10 to 40 percent (wt/wt) sucrose gradient (in SGB) and
centrifuged at 24,000 rpm for 30 minutes at 23°C in a Spinco SW 50.1 rotor. Fractions
were collected and assayed for radioactivity. Some gradients also contained
C-labeled T^ bacteriophage added as a sedimentation marker. This procedure is
summarized in Figure 1.
GROWTH MEDIA
(GROWTH MEDIA FROM BOTH TRITON LYSIS
AND FREEZE-THAH PROCESSED THE SAME)
LOW-SPEED CENTRIFUGATION
(700xg 10 min)
SUPERNATANT FLUID PELLET
I (PIB)
HIGH-SPEED CENTRIFUGATION
(75,000xg 1 hr)
SUPERNATANT FLUID PELLET
(DISCARD) "FREE"
TRITON LYSIS
LOW-SPEED CENTRIFUGATION
(700xg 10 min)
-*- CELLS.
SUPERNATANT FLUID
PELLET
(PIB)
NON-OCCLUDED VIRIONS
See Figs. 3A, C
HIGH-SPEED CENTRIFUGATION
(75,000xg 1 hr)
SUPERNATANT FLUID PELLET
(DISCARD) CELL
FREEZE-THAW
1
LOW-SPEED CEIfTRIFUGATION
(700nq '0 m'n)
PELLET
(PIB)
SUPERNATANT FLUID
I
HIGH-SPEED CENTRIFUGATION
(75,000xg 1 hr)
SUPERNATANT FLUID PELLET
(DISCARD) CELL
ASSOCIATED
NON-OCCLUDED
VIRIONS
1 I
See Fig. 3D See Fig. 3B
ASSOCIATED
NON-OCCLUDED
VIRIONS
PIB POLYHEDRAL INCLUSION BODIES.
*
Growth media was removed and "free" non-occluded virions purified, as indicated in the flow diagram. In two different
experiments, cell-associated virons were extracted by either (1) lysing cells with non-ionic detergent Triton X-100,
or (2) by freezing and thawing the cells three times. Further purification was performed as indicated.
Figure 1. Purification of H-labeled A. californica virus infected T. ni cells
48 hours post-infection*
Purification of Cell-Associated, Non-Occluded -H-Labeled Virions
Non-occluded H-labeled virions were liberated from cells (after removing Hi-
labeled growth media 48 hours after infection) by two different procedures. In one,
cells were suspended in a small volume of buffer and subjected to three cycles of rapid
freezing and thawing. In the other, cells were suspended in a buffer containing the non-
ionic detergent, Triton X-100, designated "Triton lysing fluid" (TLF) (0.25 percent
Triton X-100, 0.01 M Tris, 0.01 M EDTA, pH 7.9). After swelling the nuclei for
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10 minutes at room temperature in 0.9 mJ? TLF, 0.1 m^ of 2.0 M NaC^ was added to
each petri plate (final concentration 0.2 M), and incubated another 10 minutes at room
temperature. Both the TLF and the "freeze-thaw" lysates were given a low speed
centrifugation for 10 minutes at 700 x g at 4°C. The pellets, which contain the
polyhedral inclusion bodies (PIB) and cell debris, were processed by treating with
detergents (DOC and SDS), and by successive equilibrium centrifugations in 1.0 to 2.2 M
sucrose solutions to purify the PIB (see previous reports). The supernatant solutions
were processed as described above for purification of "free" non-occluded virions. The
purification procedure is summarized in Figure 1.
INFECTION OF MONKEY KIDNEY CELLS (CV-1) WITH A. californica NPV
Monkey cells (CV-1) grown in monolayers (90 percent) confluence on petri plates
(100 cm) were infected with A. californica NPV by inoculating each plate with 1.0 mtt
O
of virus containing insect media (10 PFU/m^) diluted 1:1 in Dulbecco's MEM media.
Infections were carried out at both 28°C and 37°C. After a two-hour adsorption period,
cells were overlaid with Dulbecco's MEM supplemented with glutamine and 5 percent
horse serum (Gibco). After 24 hours, cells were scraped from some of the plates and
processed for electron microscopy. The remaining cultures were routinely subcultured
every five to six days. Prior to subculture, media was removed and stored at -20 C for
later studies of infectivity.
DETECTION OF PIB PROTEIN BY INDIRECT IMMUNOFLUORESCENT
ANTIBODY STAINING
Monkey and insect cells were prepared for fluorescent antibody staining by
seeding them in 10 x 35 mm petri plates containing glass coverslips. Cells were
infected with 0.2 mf. of inoculum as usual, and overlaid with 2 ml of media. After
48 hours, coverslips were washed three times in phosphate-buffered saline (PBS), and
fixed by adding 2 mJl of ether-ethanol (95 percent), 1:1 for 10 minutes at room
temperature. The ether-ethanol was removed and the cells fixed with 95 percent
ethanol for 20 minutes. In some cases, the cells were given a 10 minute wash with our
standard alkaline PIB dissolution buffer (0.05 M Na-CO,, 0.05 M NaCjO. Cells were
again washed in PBS, covered with 0.1 mJl of rabbit anti-A. californica NPV anti-sera
(donated by Dr. Phillip Norton) (1:10 dilution), and incubated for 60 minutes at 37°C.
Cells were washed three times in PBS and covered with 0.1 m^ of goat anti-rabbit
fluorescein-conjugated IgG (7S fraction, ICN Biochemicals) (1:10 dilution) and incubated
for 60 minutes at 37°C. Cells were washed three times in PBS and mounted face down
in glycerol-PBS (9:1) on glass slides. Slides were stored at 4°C until examination with a
fluorescence microscope.
ELECTRON MICROSCOPY
Thin Sections
Cells were centrifuged at 600 x g, washed two times in PBS, and fixed in
2 percent phosphate-buffered glutaraldehyde. The cells were suspended in 1 percent
osmium tetroxide (one hour) before dehydration and embedding. Sections were stained
with 2 percent uranyl acetate and 0.5 percent lead acetate.
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Negative Staining of Virions
Virions were fixed in 2 percent phosphate-buffered glutaraldehyde from one to
three hours, then dialyzed against distilled water. Drops were placed on paraffin blocks
and collodion carbon-coated copper grids placed face down on the drops for three
minutes. Grids were removed, and the edge touched to filter paper to remove excess
solution. They were then placed on drops of 2 percent uranyl acetate or 2 percent PTA
for one to six minutes, blotted, and dried. Grids were examined in a Hitachi HU-11
electron microscope. Magnifications were determined from a grid of a silicon-coated,
cross-hatched grating with 54,864 lines/inch.
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SECTION 5
RESULTS
DEFINING THE INFECTIOUS UNIT
Incorporation of Tritiated Thymidine into A. californica NPV
To determine the optimum labeling conditions for virus, eight infected petri plate
cultures were overlaid with radioactive thymidine (methyl- H) and incubated at 28 C.
At various times, two cultures were harvested, the growth media removed, and "free
non-occluded virus" purified (see Figure 1). The cells were lysed by repeated freeze-
thaw and the "cell-associated, non-occluded virus" purified (Figure 1). Samples were
removed and assayed for the amount of radioactivity by scintillation counting. The
results are shown in Figure 2. From this figure it can be seen that radioactivity
accumulated most rapidly between about 24 and 40 hours after infection, and thereafter
appeared to level off. Between 40 and 48 hours, the amount of virus in cell-associated
virus actually decreased, and this may be due to rapid incorporation into polyhedral
inclusion bodies. From these data it appears that between 40 to 48 hours ic the
optimum time period to harvest cells and media for the maximum incorporation of
3
H-thymidine into non-occluded virions.
Incorporation oi Various DNA Precursors Into NPV
Since it has been reported that thymidine is not efficiently incorporated into NPV
when grown in larvae, several other pyrimidine precursors of DNA were tested in tissue
culture cells to determine the most suitable precursor for H incorporation. These
were deoxycytidine, thymine, deoxycytidine-5'-monophosphate, and thymidine. Precur-
sors were added to infected cells immediately after infection, cells and media
harvested at 48 hours, and the various fractions assayed for radioactivity. The results
are summarized in Table 1. These data show that thymidine is incorporated at the
greatest rate, while deoxycytidine is incorporated at the lowest rate. Importantly, the
majority of the radioactive virus is incorporated into polyhedra, while less than
10 percent is found in the growth media. Furthermore, the amount of non-occluded,
cell-associated virus is three to five times greater than the "free" virions in the growth
media.
Velocity Sedimentation Analysis of Labeled Virions
Virions were analyzed by velocity sedimentation through 10 to 40 percent (wt/wt)
sucrose solutions. Free non-occluded virions were derived from growth media of T. ni
cells 48 hours after infection. Two procedures were used to obtain non-occluded, cell-
associated virus. In one, cells were ruptured by freezing, followed by rapid thawing. In
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IO
g
X
^
Q_
O
X
ro
5-
10 20 30 40
Hours Post Infection
Figure 2. Incorporation of H-thymidine into non-occluded A. californica NPV.
o
Following infection of T. ni cells with A. calif ornica NPV (10 /ml),
cells were grown in media containing H-thymidine (20 fxCi/m^). At
the times indicated, growth media was removed and "free" non-
occluded virions purified as indicated by the scheme shown in Figure 1.
At the same time, cell-associated non-occluded virions were purified
by lysed cells (freeze-thaw procedure, Figure 1). Ordinate scale repre-
sents acid-precipitable radioactivity. Open circles, virions in growth
media; closed circles, virions from cells.
TABLE 1. INCORPORATION OF RADIOACTIVE DNA PRECURSORS INTO
A. calif ornica NPV
Precursor
Deoxycytidine
Thymine
Deoxycytidine
Monophosphate
Thymidine
Growth Media
("Free" Non-Occluded
Petri Dish Virions)
CPM/PD
mmo1? % of Total
x 10" Incorporation
5.3 1
529 7
19,209 4
131,289
Cells
(Cell-Associated/
Non-Occluded Virions)
CPM/PD
mmole
x 10'8
18
2,809
70,326
96,747
% of Total
Incorporation
3
36
15
Polyhedra
Inclusion Bodies
CPM/PD
mmole
o
x 10'8
656
4,390
368,987
a
% of Total
Incorporation
96
57
80
Polyhedra were not quantitated in this experiment.
10
-------
the second, cells were lysed by the non-ionic detergent Triton X-100 (see Section 2).
The purification procedures are diagrammed in Figure 1.
Analysis of the virions is shown in Figure 3. The virions in growth media segment
with a velocity of about 1000 S relative to C-labeled T. bacteriophage (Figures 3a
and 3c). (NOTE: Correction is made for density differences between T^ and A.
californica NPV.) The peak of virus is found in the lower third of the gradient.
However, also evident is material sedimenting at the top of the gradient with a velocity
of less than 100 S. As shown in Figure k, this material is sensitive to deoxyribonu-
clease. Since this DNA originated from a virus pellet, it is concluded that it represents
DNA liberated from virus (see below for more details on stability of virus). Although
there are no values reported for the sedimentation rate of A. californica NPV virions,
T. ni NPV have a reported sedimentation rate of 860 S, so that the value of 1000 S for
A. californica virions is within the expected range for non-occluded virions. Concerning
the sedimentation rate for DNA, other NPV DNAs which have been studied have
molecular weights of about 100 x 10 daltons and S values of about 60 to 70 S. It is
concluded, therefore, that the purification process as performed in this work produces
intact virions, but also causes partial disruption of the virions. Experiments are now
underway to evaluate improved procedures for purification of free, non-occluded
virions.
As shown in Figure 3b, the freeze-thaw procedure for liberating virus from cells
disrupts the virions completely, and only DNA is obtained. This would be consistent
with reports that virus in growth media loses activity upon freezing and thawing and
storage at -20 C.
In contrast, lysis of cells by Triton X-100 gives the same relative proportion of
intact virions as is found in the growth media (Figure 3d). This latter procedure is thus
relatively effective in obtaining virus from cells; however, again, there is a consider-
able amount of virus which is broken down to DNA. Importantly, Triton lysis appears to
be a valuable method for extracting non-occluded virions from infected cells, since this
has not been previously reported. From our experiments, it is now known whether the
lysing process iself or subsequent purification is responsible for the certain percentage
of virus breakdown.
Physical and Chemical Composition of Virions
Triton lysates and growth media containing infectious virus were treated with
chemicals and enzymes and examined in the electron microscope to identify the
radioactively labeled virion. The results of the chemical and enzymatic treatments are
shown in Figure ^. These results are summarized as follows.
1. The fast sedimenting virion is insensitive to DNase, but the slow sedimenting
material is sensitive to the enzyme.
2. Magnesium alone has no effect on either the virion or the DNA; thus, there is
evidently no virion-associated nuclease.
3. Deoxycholate (DOC) degraded the virion. A small intermediate peak with an
S value of about 300 S, nevertheless, is generated.
11
-------
4. The ionic detergent sodium dodecyl sulfate (SDS) degrades the virion to free
DNA.
5. Virions are insensitive to Triton X-100.
(It is important to note that the 1000 S peak is defined here as the non-occluded
virion.)
15
10
2
D_
O
15
10
(a)
(b)
<*f-r i
10 20 30 10
Fraction Number
20 30
Figure 3. Velocity sedimentation of virions in sucrose gradients. H-labeled virions
purified as indicated by Figure 1 were concentrated, layered onto 5.0 m
10 to 40% sucrose gradients and centrifuged for 30 minutes at 24,000 rpm
in a Spinco SW50.1 rotor at 23 C. Fractions were collected and acid-
precipitable radioactivity determined. Growth media (free non-occluded
virus) was removed from cells before lysis by either (a) freeze-thaw cycles,
or (c) Triton. Cell-associated virus was processed by either (b) freeze-
thaw cycles, or (d) Triton lysis. Arrows indicate the position of C-
labeled T. bacteriophage added as a marker. Sedimentation is from right
to left. *
12
-------
10 20 30
10 20 30
Fraction Number
10 20 30
Figure 4. Velocity sedimentation analysis of virions treated with DNase and
detergents. Virions were prepared from cells labeled with H-
thymidine and harvested 48 hours after infection. Virions were
incubated for 30 minutes in the presence of the detergents or DNase
at 37 C and then sedimented as indicated in the legend to Figure 3.
Concentrations used were 0.5% DOC, 1% SDS, 1% Triton, 10 p,g/mi
DNase and 0.01 M Mg++. lifC-labeled T. bacteriophage was added
as a marker (open circles).
DOC and SDS sensitivity suggests the virion is associated with membranous
material, although DOC can also dissociate certain DNA-protein complexes. Triton is
known to solubilize cytoplasmic membranes, but nuclear-type membranes are not
degraded by this detergent. Perhaps most significant is the fact that the virus,
although multiply embedded in polyhedra, is found as a single sedimenting peak in these
gradients.
Electron Microscopy of Virions
Peak fractions from sucrose gradients were fixed in glutaraldehyde and virions
examined by negative staining with phosphotungstic acid (PTA), or with uranyl acetate
(see page 7, Negative Staining of Virions).
The particles from the sucrose gradients appear as rod-shaped "bags," with an
average length of about 317 nm and a diameter between about 40 and 80 nm with PTA
13
-------
staining (see Figure 5). These dimensions can be compared with the occluded dark-
staining virus rods observed in sections of polyhedra, which are about 240 to 270 nm
(see Figure 6). However, if the outer dimensions of a single occluded rod plus the light
staining membranes are taken, this value is about 315 nm (Figure 6); i.e., the same as
the length of the free non-occluded rods. Thus, the virus "bags" prepared from sucrose
gradients correspond in size to a single rod surrounded by a membrane.
Several other important structures are seen by electron microscopy. At each end
of the rods are "end plates" which appear to contain a number of spikes (Figure 5).
These end plates are encased in caps which occasionally can be seen to be hinged or
ripped out. Also seen are a number of empty "bags" or "ghosts." These empty "bags,"
as well as full particles, can apparently polymerize end-to-end (Figure 5). Surprisingly,
even after fixation, disrupted particles and empty "bags" can be seen. It thus appears
that free virions are extremely sensitive to osmotic and/or shear forces, which may
explain the reason for the large amount of viral DNA seen in sucrose gradients.
In these electron micrographs, no structure corresponding to the inner dark-
staining rod observed in sections of polyhedra is found. Explanation for this may be
that the "inner" rods are extremely labile. Actually, virus observed by uranyl acetate
staining show "bags" which appear to be "exploding" at both ends (Figures 7 and 8).
Within these "exploding" particles are amorphous structures made up of hollow sub-units
about 15 nm in diameter. It can be speculated that the inner rods are formed by
polymerization of these sub-units.
Inf activity of Virions
Labeled virions were added to both T. ni and monkey kidney cells, and the uptake
of radioactive virus monitored. However, the amount of radioactivity was too low to
arrive at meaningful conclusions as to the absolute uptake of radioactivity. These
experiments are being repeated with virus labeled to very high specific radioactivity.
The specific infectivity of these virions is also being determined.
Virions Derived From Polyhedra as Infectious Units
Since "free" and "cell-associated" non-occluded virions only account for about 10
to 30 percent of the total labeled virus in cell cultures (the majority of virions having
been occluded into polyhedra), attempts were made to obtain virions from polyhedra by
alkaline dissolution. The results of these experiments are shown in Figure 9. The top
two panels (Figures 9a and 9b) represent velocity sedimentation in 10 to 40 percent
sucrose gradients designed to detect non-occluded virions, while the bottom panels
(Figures 9c and 9d) represent equilibrium sedimentation in sucrose (1 to 2.2 M). In the
left panels (Figures 9a and 9c), alkali degradation was performed in the presence of the
inhibitor of proteases PMSF (phenyl methyl sulfonyl fluoride), since it has recently been
reported that polyhedra contain an endogenous protease. However, in all cases, no
peaks corresponding to intact virions were found upon alkaline degradation of the
polyhedra. At this time, the explanation for failure to obtain virions from polyhedra is
not known.
-------
X 133,000
Figure 5. Particles from sucrose gradients (rod-shaped "bags") with an average
length of about 317 nm and a diameter between about *0 and 80 nm
with PTA staining
15
-------
Figure 6. Occluded dark-staining virus rods in sections of polyhedra which are about
240 to 270 nm
16
-------
X 133,000
Figure 7-
show
17
-------
Figure 8. Virus observed by uranyl acetate staining show "bags" which appear
to be "exploding" at both ends
18
-------
Q.
O
10
20 30 10
Fraction Number
30
Figure 9. Alkaline degradation of H-thymidine labeled A. californica polyhedral
inclusion bodies. Polyhedral inclusion bodies were dissolved in pH 10.5
phosphate buffer in the presence (a and c) or absence (b and d) of the
protease inhibitor phenyl methyl sulfonile fluoride (0.2%) for two hours
at 37 C. Samples were analyzed by velocity sedimentation of 10 to 40%
sucrose gradients (a and b) or by equilibrium sedimentation in 1.0 to 2.2 M
sucrose (c and d). Arrows and brackets indicate the position of untreated
virions and polyhedral bodies, respectively.
INFECTION OF MONKEY CELLS WITH A. californica NPV
Morphology of Infected Cells
Monkey kidney cells (CV-1) were infected with A. californica NPV at 28°C and
37 C (see Section 2). Cells were incubated for 24 and 48 hours, and cells harvested for
examination by electron microscopy. Cells were either scraped from the petri plates or
trypsinized to remove them.
Electron microscopy failed to reveal any structures which resembled virions or
polyhedra, nor was there any evidence that virions had penetrated the cell membrane.
Although the CV-1 cells did contain occasional inclusion bodies, none of these were
19
-------
observed in nuclei or had the overall dimensions (see Figure 6) or morphological
characteristics typical of polyhedra (Figures 10 and 11).
Other cultures were maintained for five or six days, then subcultured by
trypsinization. To date, no morphological difference has been detected between
infected cells and control uninfected cells propagated alongside the infected cells.
Repeated examinations by phase and electron microscopy have been carried out.
Monkey cells maintained at 28°C grew very slowly and assumed a bizarre
morphology (Figure 12); this was characteristic of both uninfected and infected cells.
After three passages and six weeks at 28 C, the cells ceased to divide, and were then
placed in the 37°C incubator. After a few days at 37 C, the morphology again changed;
this time the cells assumed a morphology similar to nerve cells. A large number of
inclusion bodies were seen in the cytoplasm of these cells. Unfortunately, control
uninfected cells became contaminated, and it is not known whether the cytoplasmic
inclusion bodies are a result of virus infection or simply an effect of temperature.
These cells are being carried and will be examined by electron microscopy.
Attempts to Detect Virus-Specific Protein in Infected Monkey
Cells by Immunofluorescence
Infected cells were tested for the presence of virus-specific protein using the
indirect immunofluorescent staining procedure. For this procedure, antibody is made in
rabbits against the purified polyhedral matrix protein (in this study, antibody was
provided by Dr. Phillip Norton, University of Connecticut, Storrs). This anti-serum is
allowed to react with cells, then fluorescein-labeled anti-rabbit goat immunoglobulin is
added to "stain" the antigen-antibody complex. Fluorescent areas within cells are
indicative of the presence of viral antigen.
When A. californica NPV anti-serum was reacted with infected CV-1 cells, no
immunofluorescence could be detected. In contrast, infected T. ni cells show very
bright fluorescence in nuclei corresponding to the polyhedral inclusion bodies (Fig-
ure 13), as opposed to the control (Figure 14).
PRELIMINARY SUMMARY
Summarizing the work thus far, it has been shown that an infectious form of the
virus, A. californica NPV, could be obtained from the growth media taken from infected
insect cells of Tricoplusia ni in culture. This virus was concentrated by centrifugation
and purified by velocity sedimentation in sucrose gradients. The virus was identified by
its morphology in the electron microscope, sedimentation properties, and infectivity.
Growth media containing free infectious virions was added to CV-1 monkey cells,
and the cells observed for signs of cytopathic effect. Observations of cytopathic effect
(CPE) were made by phase contrast microscopy as well as by electron microscopy.
Infected monkey cells were further tested for the presence of viral matrix protein by
indirect immunofluorescence, using antibody prepared in rabbits. Tests for the
persistence of virus within the monkey cells were uniformly negative.
20
-------
10,000 A
Figure 10. Morphological characteristics typical of polyhedra
21
-------
* '
•Tpm., ,, , ~m* *.*
.»•*,» * >» '*>«*% ~ s
^* * Jf
t * *C ^*
-/«
«* ••*# ^*
flN^'-. * *f«*,t
%». i«5*
'*•»•
*»
*«
r*
ft
*»
Figure 11. Morphological characteristics typical of polyhedra
22
-------
10,000 A
'
Figure 12. Bizarre morphology of monkey cells maintained at 28°C
23
-------
Figure 13. Fluorescence in nuclei of infected T. ni cells
Figure 14. Control for T. ni cell fluorescence experiment
2/4
-------
These experiments, however, did not provide any information as to the fate of the
infecting virus particles or the viral nucleic acid. Rather, they were designed to test
for gross pathologic changes which might be induced by the insect virus.
It was.therefore of interest to investigate the possibility that the virus might have
adsorbed to the cells, penetrated the cells, and thus able to persist in an "occult" or
latent form. This is crucial to the question of whether the virus represents a possible
biohazard since in such cases of latent persistence, possible activation of virus has been
known to result in an overt pathology.
25
-------
SECTION 6
TO DETERMINE WHETHER FREE, NON-OCCLUDED AUTQGRAPHICA CALIFQRNICA
NUCLEAR POLYHEDROSIS VIRUS CAN ADSORB AND PENETRATE
MAMMALIAN CELLS IN VITRO
The experiments described below were designed to answer the following questions:
1. Do virus particles of A. californica NPV adsorb and/or penetrate CV-i
monkey cells?
2. Does the viral nucleic acid of A. californica NPV persist in the monkey cells
throughout several cell divisions?
Virus was prepared which had a radioactive label incorporated into its nucleic
acid. This radioactive virus was used to infect monkey cells. The penetration of the
virus into the cells was monitored by determining the quantity of radioactivity taken up
by the cells. To ascertain the fate of the infecting nucleic acid, cells were lysed, and
the nucleic acid extracted and analyzed by density gradient sedimentation. In the
following sections these experiments are described in detail.
PREPARATION OF 3H-LABELED FREE NON-OCCLUDED A, CALIFORNICA VIRIONS
IN T. ni CELLS IN VITRO (USED FOR INFECTION OF MONKEY CELLS)
Cell monolayers of T. ni were infected as described previously. The infected cells
were overlaid with media containing 50 pCi/mlt of H-thymidine and incubated
48 hours at 28°C. Growth media was removed and centrifuged at 700 x g for
10 minutes at 4 C. The resulting pellet (polyhedra and cell debris) was combined with
the cells.
The supernatant (growth media containing infectious free non-occluded NPV) was
centrifuged at 27,000 rpm for 60 minutes to concentrate the virions. The virus pellet
was resuspended to 1.0 mH of Tris-NaC^ buffer (0.05 M Tris, 0.3 M NaCj? pH 7.5).
Analysis of the concentrated free virions by velocity sedimentation in 10 to 40 percent
sucrose gradients is shown in Figure 15. In this figure the free virions are shown to be
sedimenting at about the same rate as C-labeled T^ bacteriophage, added here as a
marker. (Note: When extrapolated to zero sucrose concentration, T, sediments at
about 700 S. Since it is known that the A. cal. virions are much less dense than T., the
A. cal. standard sedimentation rate would be much greater, i.e., about 1000 S.) The
peak at the top of the gradient is free DNA. It is not known how much of this DNA
represents contamination due to breakdown of cellular DNA.
26
-------
2 —
1 —
10
RT-15332
20
FRACTION NUMBER
Figure 15. Velocity sedimentation of A. cal. virions in a sucrose gradient. H-labeled
A. cal. non-occluded NPV was concentrated by sedimentation and resus-
pended in Tris-NaC-f buffer, pH 7.5 (see text). An aliquot was removed,
14
mixed with C-labeled T. bacteriophage (added as a marker) and layered
onto a 5 mi. 10 to 40% (wt/wt) sucrose gradient. The sucrose gradient was
centrifuged for 30 minutes at 24,000 rpm at 23°C in an SW50.1 rotor.
Fractions were collected and assayed for TCA-precipitable radioactivity.
To purify non-occluded virions for infection of monkey kidney cells, the remainder
of the concentrated virion preparation was layered onto a similar 10 to 40 percent
sucrose gradient and centrifuged as above (see Figure 16). The fractions containing
labeled virions (//14-//22) were pooled and dialyzed into phosphate buffered saline for
subsequent infection. The final preparation was characterized by an absorbance
spectrum ratio at 260/280 nm of 1.59, and contained about 500,000 cpm.
27
-------
A. cal. INFECTED T. ni CELLS
GROWTH MEDIA
TOO x g
10 MINUTES
CELLS
-(CONTAIN PIB'S AND
CELL-ASSOCIATED
VIRIONS)
SUPERNATANT
FLUID
PELLET
(CELLS AND DEBRIS)
PELLET RESUSPENDED
IN 1.0 m£ Tris-HCS.
BUFFER
27,000 rpm
60 MINUTES
SUPERNATANT FLUID DISCARDED
CENTRIFUGATION IN A 10 TO 40% SUCROSE
GRADIENT, 30 MINUTES 24 K
FRACTIONS CONTAINING VIRIONS
POOLED AND DIALYZED INTO PBS
RT-15328
Figure 16. Purification of H-labeled A. cal. non-occluded NPV
INFECTION OF MONKEY KIDNEY CELL CULTURES WITH
A. cal. NON-OCCLUDED VIRIONS
'H-LABELED
Small petri plate cultures (2.0 cm) containing either CV-1 cells (monkey kidney)
or T. ni cells were prepared by mixing 0.2 mi of virus in PBS (80,000 cpm) with 0.1 m^
of Mink's growth media (TNM-FH). Cells were incubated either 2, 4, or 6 hours (CV-1
cells were placed in a 5 percent CO~ atmosphere). At each of these times, the inocula
were withdrawn and counted in a liquid scintillation counter. (Prior to counting, the
samples were precipitated with TCA and washed with alcohol to remove any acid
soluble material.)
The results are presented in Figure 17. During the first two hours of infection,
about 60 percent of the A. cal. virus was absorbed by the T. ni cells. Surprisingly, CV-1
cells adsorbed a similar or even greater percentage of the infecting virus. This is
28
-------
remarkable when it is considered that no cytopathic effect was detected by conven-
tional methods. Further incubation of CV-1 cells with virus resulted in the adsorption
of about an additional 20 percent over the next four hours (Figure 16).
o
cc.
40 —
20 —
RT-15329
246
TIME (HOURS AFTER INFECTION)
Figure 17. Uptake of radioactive A. cal. virions by CV-1 monkey cells and T. ni
insect cells. Purified H-labeled A. cal. virions (80,000 cpm/dish)
(see text) were added to either CV-1 or T. ni cells and allowed to
adsorb at 28°C. At 2, 4, and 6 hours, growth media was removed,
centrifuged to remove any unattached cells, and samples of the media
were counted in a scintillation counter. The percentage of radioac-
tivity remaining in growth media is plotted against time after infection
In an attempt to determine whether the virus was loosely associated with the cells
or bound in a stable association, the cells were washed three times with PBS, then lysed
with 0.2 mj? of 0.6 percent SDS (in 0.01 M EDTA pH 7.5). The resulting lysate was
layered onto a 5 m^ 5 to 20 percent sucrose gradient (w/w) and centrifuged at
40,000 rpm for one hour in an SW56 swinging bucket rotor at room temperature.
The results of this experiment are shown in Figure 18. This figure shows the
sedimentation pattern of viral DNA in SDS lysates prepared from CV-1 infected
29
-------
cultures six hours following infection. Even after six hours, it can be observed that
most of the viral DNA sediments as high molecular species. About 20 percent of the
DNA is found sedimenting at a rate comparable to intact DNA, while the remainder
sediments at rates corresponding to breakdown products. These breakdown products
represent molecules that were generated by introducing from 1 to about 9 double-
stranded breaks in the native molecule. Interestingly, this type of partial degradation is
also found in lysates of infected T. ni cells (data not shown).
4 -
RT-15331
10 20
FRACTION NUMBER
30
40
Figure 18. Sedimentation of an SDS lysate of CV-1 monkey cells six hours following
infection by H-labeled A. cal. virions. Six hours following infection of
CV-1 cells with H-labeled A. cal. virions (80,000 cpm/dish), cells were
washed three times with PBS and 0.2 mi of SDS added to each petri dish.
The resulting lysate was gently poured from the dish (to avoid shearing
the DNA) and the lysate placed onto a 5 ml 5 to 20% sucrose gradient.
The gradient was centrifuged for one hour at 40,000 rpm in a Spinco
SW56 rotor. Centrifugation was from right to left.
In order to determine how much of the total adsorbed DNA this represents, the
radioactivity in the gradients was summed and corrected for quench. The results are
summarized in Table 2. Lysates prepared from CV-1 cells four hours after infection
reveal that at least 79 percent of the adsorbed virus can be accounted for, while after
six hours this figure is about 52 percent. Thus, it appears that most of the virus which
30
-------
adsorbs to the monkey cells remains associated with the cells even after washes.
Furthermore, the DNA persists in high molecular weight species. The fact that the
DNA remains in a high molecular weight form suggests that this DNA could conceivably
be transcribed into viral gene products. However, it should be pointed out that these
experiments as performed above are unable to distinguish between adsorbed virus and
intracellular virus.
TABLE 2. UPTAKE OF RADIOACTIVE A. cal. VIRIONS BY T. ni CELLS
Cell
Type
CV-1
T. ni
CV-1
T. ni
CV-1
T. ni
Time
After
Infection
2 hours
2 hours
hours
6 hours
6 hours
Radioactivity3
Not Adsorbed
29,500
35,500
16,300
28,600
10,000
18,800
Maximum
Radioactivity
Adsorbed
50,500
ff ,500
63,700
51, WO
70,000
61,200
Radioactivity as
Cell-Associated
DNA
34,588
50,552
36,098
Radioactivity in growth media removed from cells after infection.
Radioactivity not adsorbed and assumed to be associated with cells. Sampling of
washes showed little elution of virus.
"Radioactivity in SDS lysates sedimenting as TCA precipitable DNA (see Figure 18).
PERSISTENCE OF A. californica DNA IN INFECTED CV-1 MONKEY CELLS
The experiments presented in the previous section provide evidence for the
adsorption and possible entry of A. cal. virions in CV-1 monkey cells. Nonetheless,
earlier experiments (see subsection 4.2) failed to reveal a cytopathology in the infected
monkey cells.
To determine whether A. cal. viral genes were able to persist in the monkey cells,
infected cells were cultured for several passages, and the DNA extracted and
concentrated. Also, DNA was extracted from A. cal. PIB's and labeled in vitro using a
"nick-repair" method employing E. coli DNA polymerase. The purpose of these
procedures was to make labeled viral DNA to use as a probe in DNA-DNA hybridization
with cellular DNA. Detection of hybrids could indicate persistence of viral genes in the
infected cells. The details of these experiments are presented in the following sections.
Extraction of DNA From Infected Monkey Cells
DNA was extracted and purified from 100 to 200 petri plate cultures of infected
or control, uninfected monkey cells. This was done in batches of 50 plates each in the
following manner:
1. Cells were lysed on the petri plates by adding 1 ml of a solution containing
8 M urea, 1 percent SDS, 10"3 M EDTA, 0.24 M sodium phosphate pH 6.8.
31
-------
2. The lysate was homogenized in a commercial Waring blender 3 x 20 seconds
at top speed. Prior to shearing, 2 ml of isoamyl alcohol was added per 50 m£
of lysate to reduce foaming.
3. The lysate was added to a column of hydroxyapatite (HA) (prepared by
packing approximately three grams of powdered HA into a 1 x 30 column
equilibrated with buffer similar to the lysing buffer, except without SDS).
Under these conditions, double-stranded DNA sticks to the column while
single-stranded DNA, RNA, and protein are eluted.
The column is washed with 50 iruC of a buffer containing 8 M urea, 0.24
sodium phosphate, pH 6.8, then with 100 ml of 0.14 M sodium phosphate
buffer pH 6.8. The absorbancy at 260 nm is monitored, and if the A26Q is less
than 0.1, the DNA is eluted. If it is not below 0.1, additional 0.14 M
phosphate buffer is added.
DNA is eluted by adding 0.48 M phosphate buffer, pH 6.8, and is collected in
3 mjC aliquots. In a typical purification, the DNA elutes in eight 3 ml
fractions. DNA eluted from this column is generally contaminated with
protein, as indicated by a 260/180 nm ratio less than 1.8. The DNA is
therefore treated with pronase (100 ^g/ml for one hour at 37 C), and
extracted with an equal volume of phenol (saturated with 0.2 M Tris, pH 7.5
buffer). The aqueous phase is diluted with water to give a final phosphate
concentration of 0.14 M, and then added to a second HA column maintained
at 60°C. This column is washed with 100 to 200 ml of 0.14 M phosphate
buffer, and the DNA eluted with 0.48 M phosphate buffer as described
earlier. DNA from this second column is generally clean, as indicated by a
260/280 nm ratio of about 1.85 and by protein and RNA analysis of the DNA.
4. DNA is then sheared to a piece size of about 400 to 600 nucleotides. This is
done in a Sorvall Ribi cell fractionator at a force of 50,000 pounds per square
inch at 4°C. After shearing, the DNA is concentrated and purified again by
passage through an HA column at 60 C.
5. The DNA is then dialyzed extensively against 5 x 10" M EDTA for three
days. Following dialysis, the DNA is lyophilized and stored at -20 C.
Extraction of DNA from T. ni Cells
DNA was extracted from A. cal. infected and control, uninfected T. ni cells by a
procedure similar to that described above for CV-1 cells. Generally, cell DNA was
relatively free of contaminating protein after a single passage through an HA column
(8 M urea, 1 percent SDS, 0.24 M phosphate buffer used to lyse the cells). Thus, the
pronase treatment and phenol extraction steps were omitted and the DNA was simply
passed through a second HA column at 60 C, sheared in the cell fractionator,
concentrated on an HA column, dialyzed and lyophilized.
Extraction of DNA from Heliothis zea Cells
A Heliothis zea cell line obtained from Dr. Timothy Kurtti was routinely
propagated in the laboratory. DNA was extracted from these cells to be used as a
32
-------
heterologous DNA control in hybridization experiments. The extraction procedure used
was similar to that described above for T. ni cell DNA.
Calf Thymus DNA
Calf thymus DNA was obtained commercially from the Sigma Chemical Company.
However, it was found to be highly contaminated with protein and RNA. Therefore,
this DNA had to be further purified. Commercial, lyophilized calf thymus DNA was
hydrated for two days in phosphate buffer (0.14 M, pH 6.8). Thereafter, it was sheared
in a Waring blender, treated with pronase, RNase, and purified by passage through an
HA column at 60 C. Further purification steps were as described for the DNA's above.
DNA Purity
To test the purities of the DNA's prepared above, each was thermally denatured in
a Gilford spectrophotometer equipped with automatic accessories for thermal denatura-
tion studies. In each case, a sample of the DNA was removed before the shearing step,
dialyzed extensively against 0.15 M NaC^, 0.015 M sodium citrate buffer pH 7, and
denatured by heating to 100°C at increments of 0.5 degrees per minute between 50
and 100°C.
Table 3 summarizes the properties of the cellular DNA's prepared for the
hybridization studies.
TABLE 3. PROPERTIES OF CELLULAR DNA PREPARED FROM A. cal. NPV
INFECTED AND UNINFECTED CELLS
Source
of DNA
T. ni
|-1. zea
CV-1 Uninfected
A260/A280
1.90
2.03
1.81
Total
Amount Hyperchromicity
(mg) Tm (%)
1.52 84.8 31.2
1.97 85.2 33.7
0.64 87.0 32.1
PREPARATION OF IN VITRO LABELED A. cal. NPV DNA
Purification of A. caL NPV PIB's from A. cal. Larvae
Initial attempts to obtain NPV DNA from infected tissue culture T. ni cells for
use as a substrate for E. coli DNA polymerase were unsuccessful for a number of
reasons. First and foremost was the fact that a very large amount of infected culture
cells yielded only a very small amount of DNA. Secondly, attempts to separate PIB's
from cellular components resulted in a loss of yield of PIB's. Therefore, infected A.
cal. larvae were used as a source of PIB's, which in turn were extracted to obtain DNA.
The infected larvae were supplied to us by Dr. Vail of the USDA, Phoenix, Arizona. The
growth of these larvae took several weeks, and, in all, several thousand larvae were
9
provided as a dried powder at 4.64 x 10 PIB's/gram.
33
-------
For purification of A. cal. PIB's, four grams of powder were weighed out and
hydrated in 100 mH of 0.05 M KCi, 0.05 M Tris buffer pH 7.5. After stirring overnight
at 4°C, the PIB's were washed four times by centrifugation at 8,000 rpm for 30 minutes
in a Beckman 321 centrifuge and resuspension of the PIB pellet in 50 mH. of buffer.
Washing was stopped when the supernatant was clear and free of visible debris. The
washed PIB's were then placed on sucrose gradients formed in steps in centrifuge tubes
by the following procedure:
15 ml 2.2 M sucrose
10 mf. 1.625 M sucrose
10 mi 1.0 M sucrose
10 mi PIB
These gradients were centrifuged to equilibrium at 11,000 rpm in the JS-13 rotor for
120 minutes.
Three thick, opalescent bands were obtained, one at the interface between each
of the sucrose density steps. The band between the 2.2 and 1.625 M sucrose contained
mostly PIB's, whereas the top two bands (the one between 1.625 and 1.0 M sucrose and
the one between 1.0 M sucrose and buffer) contained mostly debris. The bottom PIB
band was collected, diluted in distilled water (1:3), centrifuged to concentrate the PIB's,
and the equilibrium centrifugation step repeated three more times. After the second
equilibrium, only a bottom PIB band was found. The top two bands were combined,
concentrated, and treated with 1 percent SDS at 50°C for one hour. Subsequently, the
remaining PIB's were concentrated and centrifuged to equilibrium several times, as
described above.
Extraction of A. cal. DNA from Purified PIB's
A number of different methods were attempted for the extraction of native high-
molecular-weight DNA from A. cal. PIB's. The first method is based on the procedure
of Gaff or d and Randall as modified by Summers and Anderson (Ref 18). Purified
polyhedral bodies were made alkaline in the presence of sodium lauryl sarcosine (0.05 M
Na2C03, 0.02 M NaCi, 10"3 M Tris, 10"* M EDTA, 2 percent SLS). The virus solution
was incubated at 60 C for 30 minutes. After cooling to room temperature, the solution
was titrated to neutrality with 1.7 N acetic acid. The solution was then extracted four
times with chloroform:butanol (3:1). During this extraction, as well as during all other
procedures, the DNA was handled very gently to avoid breaking the DNA by shear. The
aqueous phase was removed and two volumes of alcohol added. The DNA was spooled
from solution on a glass rod at the interface between the aqueous and alcohol. The
DNA was then dissolved in 0.015 M NaCi, 0.-015 M sodium citrate overnight. The
260/280 ratio of this DNA was 1.87- This DNA was subsequently phenol-extracted, but
the 260/280 ratio did not increase.
The second method used was found to be simpler and yielded high molecular-
weight DNA. This is the method adopted for routine extraction of A. cal. NPV DNA.
Purified polyhedral bodies were mixed with an equal volume of 0.2 M Na-PO., pH 10.5
for 20 minutes, by which time the reaction mixture had cleared. SDS was added to a
final concentration of 0.2 percent and the solution incubated at 50°C for 30 minutes.
-------
Thereafter, the lysed virus was placed onto a 5 to 20 percent (wt/wt) sucrose gradient
and centrifuged at 40,000 rpm for 60 minutes at 23°C in an SW56 rotor. The resultant
gradient showing 3H-labeled A. cal. DNA with 1IfC-labeled SV40 DNA as a marker is
shown in Figure 19. The resulting peak of A. cal. DNA is monodispersed and sediments
at about 50 S. This DNA peak was pooled and dialyzed against 0.01 M Tris, 10 EDTA
pH 7.5. For preparing DNA for hybridization, a similar procedure was followed using
PIB's obtained from larvae (see above), and this DNA was used as a template for E. coli
polymerase in the synthesis of a labeled probe. In sucrose gradients, the DNA peak was
monitored by determining the absorbance at 260 nm.
15 -
o
X
Q.
O
10
RT-15330
10 20 30
FRACTION NUMBER
40
Figure 19. Sedimentation of A. cal. NPV DNA extracted by alkali and SDS from
purified PIB. H-labeled A. cal. PIB purified from infected T. ni
cells was extracted by treatment with SDS at pH 10.5, as described
in the text. The polyhedral lysate was mixed with C-labeled SV40
DNA added as a marker and sedimented at 40,000 rpm for 60 minutes
at 23 C in a Spinco SW56 rotor. Radioactivity represents TCA pre-
cipitable counts.
IN VITRO LABELING OF A. cal. NPV DNA EMPLOYING E. coli DNA POLYMERASE
The following method for the in vitro labeling of DNA is a modification of the
method first described by Nonoyama and coworkers (Ref 19) and modified by
Drs. Wayne Lancaster and William Meinke of Scripps Clinic. High molecular weight
native, double-stranded DNA is reacted with low concentrations of deoxyribonuclease
to introduce single-stranded nicks into the DNA. This first reaction is stopped, and the
nicked DNA is then reacted with E. coli DNA polymerase under conditions during which
35
-------
the enzyme simultaneously digests the DNA (5'-end) and then synthesizes complemen-
tary base sequences from the 3'-end (translation reaction). By employing radioactively
labeled deoxyribonucleotide triphosphate precursors, the labeled bases are incorporated
in short stretches. In this way, labeled DNA can be obtained with specific activities of
several million cpm/ptg of DNA. A typical experiment is described as follows.
Nicking Reaction (0.642 ml)
5 \iglmH DNA in
0.03 M Tris, pH 7.5
0.03 M KCl
0.003 M MgCj?2
0.01 pig DNAse
(Ratio of DNA to DNAse 500/1, wt/wt)
The mixture is incubated for either 12 or 24 minutes at 37 C
The reaction is stopped by heating to 65 C for five minutes.
Translation Reaction (50 p. f )
5 x 10"2 ng DNA*
5 x 10~2 M
5x 10"3 M
_3
5 x 10 M /3-mercaptoethanol
3 x 5 x 10 deoxynucleotide triphosphates (dCTP, dGTP, dATP)
10 MCi dTTP -» 3H (NEN)
5 pi of DNA polymerase (Bohringer-Mannheim).
Mixture was incubated for 30 minutes at 20°C.
Reaction stopped by heating at 68°C for five minutes.
Purification of the Labeled DNA
In order to remove the unreacted bases and label, the reaction mixture was passed
over a G50 Sephadex column equilibrated with 0.02 M Tris, pH 8, 10 M EDTA and
0.1 percent Sarkosyl; 50 [iH was applied to a column, 0.7 x 22 cm. The labeled DNA was
eluted in a void volume, while the low molecular material was retarded. The labeled
DNA was pooled and counted in a liquid scintillation counter. The size of the DNA was
determined by alkali sucrose gradient centrifugation. The reason for this latter
*
Reactions carried out using DNA that was nicked for either 12 or 24 minutes (see
above).
36
-------
procedure was that there is an optimum piece size for reassociation, and this is between
200 and 600 nucleotide base pairs. Thus, the analysis by alkaline sucrose density
gradient centrifugation was a monitor of the extent of the nicking reaction, as well as a
test of the extent of nuclease versus polymerase in the translation reaction.
In the alkaline gradient, a 7 S lifC-labeled human cellular DNA was used as a
marker. The results of this nick-translation are shown in Table <4. With both a 12- and
24- minute incubation, the amount of incorporation into A. cal. was about
1 x 10 cpm/ng. This is exceedingly high compared to in vivo labeling; however, it is
only about 15 percent of the optimum amount of incorporation usually obtained by this
method using small molecular-weight viral DNA's such as SV40 DNA or human
papilloma DNA. Furthermore, the size of the DNA was found to be too large for
optimum hybridization. This would suggest that the nicking times and/or deoxyribonu-
clease concentrations must be changed. Experiments are presently in progress to
improve the efficiency of incorporation, as well as to adjust reaction conditions in order
to generate a labeled product with the properties of a suitable probe.
TABLE 4. NICK-TRANSLATION OF A. cal. DNA BY
E.coli DNA POLYMERASE
DNase Specific Sedimentation
Treatment Activity Rate of DNA
Time of DNA in Alkali
(minutes) (cpm/fjg) S (Svedbergs)
12 1,200,000 >10
2k 1,000,000 >10
37
-------
SECTION 7
THE USE OF DNA-DNA HYBRIDIZATION FOR DETECTION OF THE
PERSISTENCE OF VIRAL GENOMES
ISOLATION OF VIRAL DNA
Characterization of In Vivo Labeled DNA
A. cal. NPV was grown in T. ni cells in the presence of H-thymidine, and virions
were isolated from the supernatant medium by centrifugation through linear 5-40
percent potassium tartrate gradients. Radioactively labeled viral DNA was extracted
from the purified virions by treatment with SDS and phenol. Equilibrium centrifugation
of the viral DNA preparation through cesium chloride and ethidium bromide allowed the
separation of supercoiled viral DNA and open circular and linear DNA forms (Fig-
ure 20).
Preparation of Unlabeled Viral DNA
Unlabeled viral DNA for use in the in vitro labeling procedure was extracted
directly from preparations of dried, frozen A. cal. infected larvae. Two to three grams
of the larval preparation were resuspended in 6 mJl of distilled water and brought to
0.03 M NaCO^ (pH 10.9). This solution was incubated at 50°C for ten minutes then
made to 0.01 M EDTA and 1 percent sodium lauryl sarcosinate, and incubated at 50 C
for an additional 15 minutes. Heavy debris was removed by low speed centrifugation,
and the supernatant was layered directly onto preformed CsCi/ethidium bromide
gradients (p 1.48-1.60 in 0.01 M Tris, pH 7.5, 1 mM EDTA, 200 ng/ml ethidium brom-
ide) and centrifuged at 100,000 x g for 18 hours. DNA in the preparation bound
ethidium bromide, and was visible as a thick red band near the bottom of the centrifuge
tube. This band and any pelleted material were resuspended in 2.5 mi of the above
buffer, and the solution was brought to p 1.602 by addition of CsCi. The solution was
covered with mineral oil and centrifuged at 32,000 rpm for 48 hours at 25°C in the
SW50.1 rotor. Supercoiled viral DNA and open circular and linear DNA were visible as
two distinct bands under UV light. Only supercoiled viral DNA from the bottom band
was utilized in preparing in vitro labeled DNA. This ensured that only viral DNA, and
no residual cellular DNA, would be included in the labeled probe.
PREPARATION OF IN VITRO LABELED VIRAL DNA
Viral DNA was labeled in vitro by the "nick-repair" technique of Lancaster and
Meinke (Refs 15,16) (Figure 21). This procedure allows the preparation of labeled DNA
with specific activities of 10 -10 cpm/jjg of DNA, higher than can be achieved
through in vivo labeling. This method has the added advantage of requiring only a small
amount of viral DNA (1 pig) for each reaction.
38
-------
70
50
30
2 10
x
to
FRACTION
Figure 20. CsC^-dye equilibrium centrifugation of A. caL viral DNA. CsC^(l g/W )
was dissolved in DNA solutions and ethidium bromide added to a final con-
centration of 100 mg/m^ in a volume of 3.0 mjf. The density of the solution
was adjusted to 1.5786, the solution covered with mineral oil and centrifuged
at 32,000 rpm for 48 hours at 25°C in the 50.1 rotor. Fractions were collec-
ted from the bottom of the tube directly onto filter paper discs and assayed
for radioactivity following treatment with trichloroacetic acid and washing
with 95% ethanol. The heavy peak (a) corresponds to closed supercoiled
viral DNA while the light peak (b) represents open circular and linear form.
A. cal. supercoiled DNA was treated with pancreatic deoxyribonuclease I at a
ratio of 1:100 (DNArDNase) to introduce single-stranded scissions. The nuclease was
inactivated by heating to 65 C for 19 minutes. Viral DNA was subsequently labeled by
initiating repair synthesis with the addition of Escherichia coli DNA polymerase I in the
presence of unlabeled ATP, CTP, and GTP and 3H-TTP. The reaction was held at
15-17°C to reduce the possibility of displacement synthesis. Repair synthesis was
terminated by the addition of Sarkosyl, and the DNA was separated from unincorpor-
ated nucleotides on a Sephadex G-50 column. This DNA preparation has a specific
activity of 7.5 x 10 cpm/ng DNA.
39
-------
Purified viral DNA in 0.05 M Tris-HC^, pH 7.5, 0.05 M
0.005 M ~~ ~
J
Add pancreatic deoxyribonuclease in 0.05 M Tris-HC^, pH 7.5,
0.005 M MgC^2 at a ratio of 1:500 (wt/wtRDNA to DNase).
Incubate at 37°C for 15 to 30 minutes to introduce "nicks".
Heat to 65°C for 19 minutes.
I
Dialyze "nicked" DNA against 0.07 M potassium phosphate,
.4, 0.007 M
"
I
Make DNA 0.1 ^mol 2-mercaptoethanol and 0.005 pimol with
each necessary unlabeled deoxynucleotide triphosphate. Add
labeled deoxynucleotide triphosphate.
Begin "repair" by addition of 20 units DNA polymerase and
incubate at 15-17 C for 60 minutes.
\
Reaction stopped by addition of Sarkosyl to 1%.
Pass reaction mixture through Sephadex G-50 equilibrated with
0.02 M Tris-HCi1, pH 8.0, 0.001 M EDTA, 0.1% Sarkosyl.
r
Pool labeled DNA peak.
Extract with phenol.
I
Precipitate with two volumes 95% ethanol.
Figure 21. In vitro labeling of viral DNA
40
-------
The in vitro labeled A. cal. DNA was sized by sedimentation through a 5-20 per-
cent alkaline sucrose gradient at 49,000 rpm for seven hours at 15°C in the SW50.1 rotor
(Figure 22). The DNA sedimented at about 7.5S, when compared with a 7S 14C-WiL2
DNA marker.
1600
1400
1200
^ 1000
T
~ 800
Q.
O
X
ro
600
400
200
7.S 6S
OL
O
o
100
75
50
25
20%
10 20 30 40
FR ACTION
50
5%
Figure 22. Alkaline sucrose gradient centrif ugation of in vitro labeled A. caL DNA.
DNA samples were centrifuged through 5-10% linear sucrose gradients in
0.3 N NaOH, 1.0 M NaC^, 10-3 M EDTA and 0.015% Sarkosyl in the SW56
rotor at 49,000 rpm for seven hours at 15°C. 14C WiL2 DNA (7S) was
added as a marker. Fractions were collected from the bottom of the
tube onto filter paper discs and assayed for radioactivity following treat-
ment with trichloroacetic acid.
-------
INFECTION OF CELL LINES
Vero (African green monkey kidney) cells were grown in Eagle's medium supple-
mented with 10 percent fetal calf serum and penicillin and streptomycin at 37 C in a
5 percent CO2 atmosphere. Two-hundred fifty plastic plates (100 cm) of cells were
prepared, and 200 plates were infected when the monolayers were approximately
80 percent confluent. An inoculum of 5 x 10 plaque forming units (pfu) per plate were
used for a multiplicity of infection of approximately one. The virus was allowed to
absorb at 37°C for 90 hours, then the cells from 50 plates were removed by
trypsinization, pelleted by low speed centrifugation, and frozen at -20 C. This sample
was considered the zero time infected sample. Identical fractions were harvested at 4,
24, and 48 hours after infection. The control sample (Vero cells unexposed to A. cal.
NPV) were also collected at 48 hours.
Plates of T. ni (insect cells) were prepared and infected to serve as positive
controls for the presence of viral DNA in infected cellular DNA preparations. The cells
were grown in Grace's modified medium supplemented with fetal calf serum, yeast
extract, lactalbumin hydrolysate, and penicillin and streptomycin at 26 C without
additional CO-. The plates were infected and harvested as described above. Nuclear
polyhedral inclusion bodies (PIB) were visible by 24 hours post infection.
ISOLATION OF CELLULAR DNA
Cellular DNA was extracted from the Vero or the T. ni samples by a ureaphos-
phatehydroxyapatite column procedure outlined in Figure 23 (Ref 20). The cells were
suspended in 8 M urea, 0.24 M phosphate, pH 6.8, 10 M EDTA, 1 percent SDS,
2 percent isoamyl alcohol, and lysed in a Waring blender. The solution was then applied
to a hydroxyapatite column equilibrated in 8 M urea, 0.24 M phosphate, and 1 percent
SDS. Cellular DNA bound to the column, while proteins and RNA were eluted. Urea
was removed by washing the column with 0.14 M phosphate buffer, then the DNA was
eluted with 0.48 M phosphate buffer. The DNA was further purified by treatment with
RNase and pronase, phenol extraction and application to a second hydroxyapatite
column. Approximately 1.0 mg of DNA was obtained from each 50 plate sample.
DNA REASSOCIATION
The ability of the DNA-DNA hybridization technique to detect small amounts of
viral DNA in cellular DNA preparations requires that nanogram quantities of denatured
labeled viral DNA be reassociated in the presence of milligram quantities of cellular
DNA. The kinetics of DNA-DNA reassociation follow the equation Co/C = 1 + KCot
where Co is the initial concentration of single-stranded viral DNA probe, C is the
concentration of the viral probe remaining single-stranded at time t, and K_ is the
reassociation constant. A plot of Co/C vertus ;t will result in a straight line if the
DNA-DNA reassociation reactions are following second-order kinetics. If unlabeled
viral DNA sequences are present in DNA extracted from infected cells, the reassocia-
tion rate of the probe will be increased in direct proportion to the amount of unlabeled
viral DNA present. The total amount of viral DNA present in a sample of infected
cellular DNA can be determined by the increase in the reassociation rate.
42
-------
Suspend cells in 8 M urea, 0.24 M phosphate, pH 6.8, 10~3 M EOT A,
1% SDS, 2% isoamyl alcohol.
Lyse cells in Waring blender.
I
Apply to HA column equilibrated with above solution (-EDTA and
isoamyl alcohol).
I
Wash column with urea buffer. Cellular DNA binds to the column,
RNA and protein are eluted.
I
Remove urea by washing the column with 0.1* M phosphate buffer.
I
Elute DNA with 0.48 M phosphate buffer.
I
Treat DNA with RNase (20 Mg/m^), then pronase (200 ng/mO.
Extract with phenol.
I
Pass through a second HA column, collect DNA as above.
*
Pass DNA through the Ribi @ 50JOOO psi (to give a piece size of 7S).
I
Apply DNA to a third HA column, collect as above.
I
Dialyze two days against 0.001 M EOT A, then three days against
5 x 105 M EDTA.
Lyophilize; store at -20°C.
Figure 23. Schematic of extraction of cellular DNA
-------
Figure 24 illustrates data from a DNA-DNA reassociation experiment set up to
determine the effectiveness of the in vitro labeled A. cal. DNA probe. Salmon sperm
DNA (100 (j.g) was mixed with varying concentrations of purified unlabeled A. cal. DNA
to simulate different amounts of unlabeled viral DNA in an infected cellular DNA
preparation. Labeled A. cal. DNA (5 ng) was mixed with 100 jug of the salmon
sperm/unlabeled viral DNA samples. The DNA solutions were denatured by boiling,
then allowed to reassociate at 60°C. At various times samples were removed and
applied to a hydroxyapatite column. Single-stranded DNA was eluted with 0.14 M
phosphate buffer, pH 6.8, while reassociated duplex DNA molecules were eluted with
0.48 M phosphate buffer, pH 6.8.
o
o
o
6.0
5.0
4.0
3.0
2.0
24
HOURS
(*-A) I00/ug SALMON SPERM ONA
(M) + 100 ng A-SflLDNA
(•-•) <• 200 ng A-col. DNA
Figure 2*. Hybridization of in vitro labeled A. cal. DNA. Labeled viral DNA (5 ng)
and salmon sperm DNA (100 p,g) containing either 0, 100, or 200 ng un-
labeled viral DNA were denatured then allowed to reanneal at 60 C.
Samples were removed at various times and single-stranded DNA was
separated from reassociated duplex DNA by hydroxyapatite column
chromatography.
Reassociation was linear for the first 72 hours. As expected, the rate of
reassociation of the labeled viral probe in the presence of 200 ng unlabeled A. cal. DNA
was approximately twice that of the probe in the presence of 100 ng A. cal. DNA. The
amount of hybridization continued to increase throughout 168 hours, when 83 percent
of the labeled viral DNA probe was reassociated. The departure from second-order
kinetics after 72 hours could indicate that the labeled probe was beginning to break
down with increased length of incubation. The number of viral genome equivalents
present in a sample will be determined from the differences in reassociation rates while
the reaction is following linear, second-order kinetics.
44
-------
SECTION 8
SUMMARY
• Large quantities of cellular DNA have been extracted from various control and
virus infected cultures. This DNA has been characterized, sized, and lyophilized.
• Large quantities of NPV virus* have been purified and the DNA extracted.
• The feasibility of preparing in vitro labeled A. cal. DNA and utilizing this as a
probe to follow the fate of insect virus genomes in infected cells has been
established.
• It has been shown that DNA labeled in vitro will hybridize with unlabeled A. cal.
DNA in a mixed DNA preparation and is thus a sensitive probe for the detection
of viral DNA.
-------
SECTION 9
REFERENCES
1. P. Collins, Nature 257:2-3 (1975).
2. R. F. Smith, "Baculoviruses for Insect Pest Control: Safety Considerations,"
American Society for Microbiology, pp. 9-11.
3. WHO Technical Report Series No. 531 (FAO Agricultural Studies, No. 91), Report
of the 3oint FAO/WHO Meeting on Insect Viruses, Geneve (1972) (World Health
Organization, Geneve, 1973).
4. M. H. Rogoff, "Baculoviruses for Insect Pest Control," American Society for
Microbiology, pp. 102-103.
5. C. M. Ignoffo, Ann. Y. Y. Acad. Sci. 217:141-164 (1973).
6. R. Rubinstein and A. Poison, 3. Insect. Path. 28:157-160 (1976).
7. H. L. Morton, 1. O. Moffett, and F. D. Stewart, 3. Insect. Path. 26:139-140 (1975).
8. A. H. Mclntosh, "Baculoviruses for Insect Pest Control: Safety Considerations,"
American Society for Microbiology, pp. 63-69 (1975).
9. R. H. Goodwin, 3. L. Vaughn, 3. R. Adams, and S. J. Louloudes, Pub. Ent. Soc.
Am. 9:66-72 (1973).
10. M. Himeno, F. Saki, K. Omodera, H. Nakai, T. Fukada, Y. Kawade, Virology
33:507-512 (1967).
11. C. M. Ignoffo and R. R. Rafajko, 3. Invertbr. Path. 20:321-325 (1972).
12. A. H. Mclntosh and K. Maramorosch, 3. N. Y. Entomol. Soc. 8h 175-182 (1973).
13. C. M. Ignoffo, M. Shapiro, and W. F. Hink, 3. Invert. Path. J&131-134 (1971).
14. K. N. Potter, P. Faulkner, and E. A. MacKinnon, 3. Virol. J.8:1040-1050 (1976).
15. W. D. Lancaster and W. Meinke, Nature (London) 256:434-436 (1975).
16. W. D. Lancaster, C. Olson, and W. Meinke, 3. Virol 17:824-831 (1976).
17. L. D. Gelb, D. E. Kohne, and M. A. Martin, 3. Mol. Biol. 57:129-145 (1971).
46
-------
18. M. D. Sumners and D. L. Anderson, Virology 50, pp. 459-471 (1972).
19. M. Nonoyama and 3. S. Pagano, Nature 242, pp. 44-47 (1973).
20. W. Meinke, D. A. Goldstein, and M. R. Hall, Anal. Biochem. 58:82-88 (1974).
47
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-80-026
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Investigation of the Persistence and Replication of
Nuclear Polyhedrosis Viruses in Vertebrate and Insect
Cell Cultures by the Use of Hybridization Techniques.
S. REPORT DATE
May 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William Meinke, D.A. Goldstein, Cynthia Alvidrez,
John Spizizen, C.B. Williams, and H.R. Lukens
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Scripps Clinic and Research Foundation
10666 N. Torrey Pines Road
La Jolla, California 92037
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
Contract No. 68-02-2209
12. SPONSORING AGENCV NAME AND ADORESS
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The goal of this research is to determine whether a nuclear polyhedrosis
virus (NPV) can interact with a variety of mammalian and insect cells in
vivo, producing either apparent or inapparent infection. The use of DNA-
DNA hybridization techniques to detect the presence of viral genomes in
cells will provide a sensitive method of monitoring viral persistence or
replication, even in the absence of cytopathic effects or the production
of infectious virus particles. It is proposed that the DNA-DNA hybridi-
zation technique be developed as a sensitive assay for the replication or
persistence of NPV in vitro, allowing a more effective evaluation of the
safety of these viruses as biological control agents that has been
possible employing previously established methods.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
b.lOENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Nuclear polyhedrosis virus (NPV)
Viral genomes
Cells in vivo
06 F,T
18. DISTRIBUTION STATEMEN1
RELEASE TO PUBLIC
19. SECURITY CLASS iThu Repori)
UNCLASSIFIED
21. NO. OF PAGES
48
20. SECURITY CLASS .
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