CMS-TECHNICAL IRPORMATIOM CBS
PB85-242527
Investigation on the Potential Environmental
Hazards of Pesticidal Viruses. 1. Molecular
Biology of 'Spodoptera frugiperda' Nuclear
Polyhedrosis Virus. 2. Lack of Evidence for
Possible Environmental Hazards
North Carolina Univ. at Chapel Hill
Prepared for
Health Effects Research Lab,
Research Triangle Park, NC
Jul 85
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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PB85-242527
EPA/600/1-85/018
July 1985
INVESTIGATION ON THE POTENTIAL ENVIRONMENTAL
HAZARDS OF PESTICIDAL VIRUSES
I. Molecular Biology of Spodoptera frugjperda Nuclear
Polyhedrosis Virus
II. Lack of Evidence for Possible Environmental Hazards
by Eng-Shang Huang, Lambert Loh, Yuan—Ming Wu* and Eng— Chun Mar
Cancer Research Center and Department of Medicine
and Department of Microbiology and Immunology
School of Medicine
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina 27514
*on leave from Wu-han Institute of Virology, Hu-pei, China
Grant Number: R806210
Project Officer: Clinton Kawanishi
Health Effect Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27709
This study was conducted in cooperation with U.S. Department of Agriculture,
Beltsville, MD 20705
Health Effect Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27709
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TECHNICAL REPORT DATA
(Please react Instructions on the reverse before completing}
1. REPORT NO.
EPA/600/1-85/018
2.
3. RECIPIENT'S ACCESSION NO.
5 2425277SS
4. TITLE ANO SUBTITLE
Investigation on the Potential Environmental Hazards
Pesticidal Viruses
oi
5. REPORT DATE'.
July 1985
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
E-S. Huang, L. Loh, Y-M. Wu and E-C. Mar
S. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADDRESS
Cancer Res. Center & Dept. of Med. & Dept. of
Microbiology & Immunology, School of Med., Univ. of
North'Carolina at Chapel Hill
Chapel Hill, NC
10. PROGRAM ELEMENT NO.
E104
1 1. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND AOORESS
USEPA, HERL, DBD, PTB
RTP, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Due to tne environmental ana ecoiogicai exiecrc^ m. \.\j*.j.\- -«rem.twa-.i. t/=.= >-j.
cides, the usage of insect viruses have been considered as one of the alterna-
tives for the control of agriculture insect pests. In fact in the past 3
decades, several baculoviruses have been used as viral peticides for pest
control. It has not been demonstrated to be hazardous to non-target organisms
using the classical infectivity and morphological alteration as measuring
factors, in this research project, we have further used molecular biological
approaches to characterize the molecular str»ucture of one of the insect
viruses to investigate and elucidate the possible pathogenicity.and oncogen-
icity of pesticidal viruses to human and other mammals at in vitro level.
Our study suggests that the pesticidal virus Spodoptera Frugiperda (SF)can not
productively infect human fibroblast or HEP-2 cell lines and can not induce
morphological transformation of human fibroblast.
Besides the study on the biopathology of a pesticidal virus, Spodoptera
frugiperda nuclear polyhedrosis virus (SfNPV), we have also extensively studied
the molecular structure of the genome of this virus justified on the need in
developing non-hazardous universal pesticidal viruses. The complete set of
virus DNA fragments have been cloned in pBR322 plasmid. This set of the
recombinant plasmid is now available for further gene function study.
17.
KEY WORDS ANO DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COSATl fie!d;Group
18. DISTRIBUTION STATEMENT
19. SECURITY CLASS iTIlit Reportl
21. NO. OF PAGES
64
2O. SECURITY CLASS iThit page)
22. PRICE
EPA Form 2220-1 (R«». 4-77) PREVIOUS EDITION is OBSOLETE .
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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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 complexities of environmental problems originate in the deep interdependent
relationships between the various physical and biological segments of man's natural
and social world. Solutions to these environmental problems require an integrated
program of research and development using input from a number of disciplines. The
Health Effects Research Laboratory, Research Triangle Park, NC and Cincinnati, OH
conducts a coordinated environmental health research program in toxicology, epidemi-
ology and clinical studies using human volunteer subjects. Wide ranges of pollutants
known or suspected to cause health problems are studied. The research focuses on
air pollutants, water pollutants, toxic substances, hazardous wastes, pesticides,
and non-ionizing radiation. The laboratory participates in the development and
revision of air and water quality criteria and health assessment documents on
pollutants for which regulatory actions are being considered. 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 environmental regulatory decisions involving the protection
of the health and welfare of all U.S. inhabitants.
F.Gordon Hueter.Ph.D.
Director
Health Effects Research Laboratory
iii
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ABSTRACT
Due to the environmental and ecological effects of toxic chemical
pesticides, the usage of insect viruses have been considered as one of the
alternatives for the control of agriculture insect pests. In fact in the past
3 decades, several baculoviruses have been used as viral pesticides for pest
control. It has not been demonstrated to be hazardous to non-target organisms
using the classical infectivity and morphological alteration as measuring
factors. In this research project, we have further used molecular biological
approaches to characterize the molecular structure of one of the insect
viruses to investigate and elucidate the possible patho geni city and
oncogenicity of pesticidal viruses to human and other mammals at in vitro
level. Our study suggests that the pesticidal virus Spodoptera Frugiperda
(SF) can not productively infect human fibroblast or HEP-2 cell lines and can
not induce morphological transformation of human fibroblast.
Besides the study on the biopathology of a pesticidal virus, Spodoptera
frugjperda nuclear polyhedrosis virus (SfNFV), we have also extensively
studied the molecular structure of the genome of this virus justified on the
need in developing non-hazardous universal pesticidal viruses. The complete
set of virus DNA fragments have been cloned in pBR322 pi asm id. This set of
the recombinant pi a sin id is now available for further gene function study.
This work was carried out in the Cancer Research Center and the
Department of Medicine, University of North Carolina at Chapel Hill under the
support of U.S. Environmental Protection Agency. This report is submitted in
fulfillment of Grant Number R806210 by the University of North Carolina under
the sponsorship of U.S. Environnental Protection Agency. The report covers
the period June 10, 1978 to September 10, 1981.
IV
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CONTENTS
PAGE
Foreword..... .............................................. ....
Abstract [[[ iv
Fi gures [[[ vi
Tables [[[ viii
Ac know 1 edgement ................................................ ix
1. Introduction and Background ........ .................. 1
References Cited ................. . ........... ........ 5
2. Conclusion and Recommendation .............. . ......... 7
3. Scientific Experiments and Results ........ ... ........ 8
Section A. General Methods for Puri-
fication of S.f. NPV and Viral DNA ................... 8
Section B. Analysis of the Spodoptera
f rugjperda Nuclear Polyhydrosis Virus
Genome by Restriction Endonncl ease and EM ............ 10
1. Abstract ................................... 10
2. Experimental Methods and Results ........... 10
3. References Cited ..................... ...... 17
Section C. Spodopt era f rug ipe r da
Nuclear Polyhedrosis Virus Genome: Physical
Maps for Restriction Endonucl ease s BamHI and
Hindll I ..... ......................................... 19
1. Abstract. ............ ...................... 19
2. Introduction ............................... 19
3. Materials and Methods ...................... 19
4 . Results ...... • • • • ........ .................. 22
5. Discussion ................................. 29
6. References Cited.... ....................... 32
Section D. Construction of a Cloned
Library of the Hindlll DNA Fragments of
SfNPV Genome and Mapping of DNA Fragments ........... 34
1. Abstract ............. .. .................... 34
2 . Introduction ............ . ........... . ...... 34
3. Materials and Methods ...................... 35
4. Results .................................... 36
5 . Discussion ........................ . ........ 42
6. References Cited ........................... 45
Section E. Interaction of S. f rugiperda
NPV With Various Mammalian Cell Lines In
Vitro ................................................ 47
1. Abstract .......... ................ ......... 47
2. Introduction ...................... . ........ 47
3. Materials and Methods ...................... 47
4. Results .................. .................. 49
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FIGURES
Number PAGE
Section B;
1 Microdensitometer scans of ant oradiographs of
SfNPV OH DNA cleaved by restriction enzyme 13
2 Cleavage patterns of DNA from the NC, OH, MS
and GA strains of SfNPV with restriction
endonucl eases 14
3 Electron micrograph of partially denatured
SfNPV DNA molecules. i 15
4 Histograms of denatured sites for SfNPV OH
DNA 16
Section C;
1 Cleavage patterns of SfNPV DNA by restriction
enzymes BamHI, Hindlll and EcoEI. 23
2 Aut or adio gr aph of patterns resulting from cross
hybridization between **P labelled and cold
BamHI fragments of SfNPV DNA 25
3 Autoradiograph between **P-labelled BamHI
and cold partially digested SfNPV DNA 25
4 Autoradiograph of *3P-labelled Hindlll
fragments and cold BamHI fragment of SfNPV
DNA 27
5 Physical map of SfNPV genome for BamHI (a),
and partial map (b) and complete map (c) for
Hindlll .- 30
6 Autoradiograph of *aP labelled EcoRI frag-
ment and cold Hindlll fragments of SfNPV DNA 28
7 Cleavage patterns of SfNPV DNA by Hindlll and
(Hindlll + BamHI) 30
8 Autoradiograph of **P labelled Hindlll frag-
ments and cold Hindlll and B am HI -doubl e-
digested DNA 31
9 Physical maps of the SfNPV genome for BamHI
and Hindlll . 31
VI
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PAGE
Section D:
1 Hindlll digestion pattern of SfNPV 37
2 Hybridization of cold B amHI-di ge s t e d SfNPV
DNA with o-»»P DNA of plasmid F.I.M.0 37
3 Hybridization of »»P labelled DNA of frag-
ment F,I,M and 0 to restricted SfNPV DNA 38
4 Physical maps of the SfNPV genome for endo-
nuclease Hindlll and BamHI 39
5a The cloned library of recombinant plasmids 40
5b Authentication of viral DNA inserts 41
Section E;
1 Infection of SF 140AE cells with virus from
hemolymph of SfNPV infected larvae (A) micro-
scopic view of inclusion (B) immunofluorescence 51
2 Transfection of SF 140 AE cell, WI-38 and
guinea pig fibroblast with SfNPV DNA 52
VI1
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TABLES
Number PAGE
Section C.
1 Cross-blot hybridization of » *P labelled
BamHI fragments to unlabelled BamHI partical
digestion products 27
2 Cross-blot hybridization of **P labelled
Eindlll fragments to unlabelled BamHI frag-
ments . 27
3 Cross-blot hybridization of »*P labelled
EcoEI fragments to unlabelled Hindlll frag-
ments 27
4 Molecular weights of Hindlll-BamHI double
digest fragments 30
5 Sequence homology between the Eindlll-BamEI
double digestion products and Hindlll or
Bam HI restriction fragments 30
6 Molecular weights of the BamHI, Hindlll and
EcoRI restriction fragments 31
Section D;
1 Molecular weights of viral DNA fragments
cleaved by Hindlll and recombinant clones
carrying corresponding DNA fragments 43
2 Southern blot hybridization of 32P labelled
Hindlll fragments to unlabelled BamHI fragments
and the linkage map of DNA fragments 44
viii
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ACKNOWLEDGEMENTS
The experimental research material of this work was partly provided by
Dr. Clinton Kawanishi of the U.S. Environmental Protection Agency at Research
Triangle Park, NC (for different insect cell lines and SfNPV) and Dr. John J.
Hamm of Southern Grain Insects Laboratory. The authors are indebted to both
of them. We also thank Dr. Y. S. Huang of the U.S. Environmental Protection
Agency for providing us the technical assistance and scientific advice.
Some of the experimental work reported here was carried out by Af shin
Heymandi and S.M. Huong with skill and patience and their contribution is
hereby acknowledged. The authors are also indebted to Hiss Barbara Leonard
for excellent secretarial assistance.
IX
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I. INTRODUCTION
In recent years, there has been great interest in industry and government
in searching for the possible usage of insect viruses as an alternative in
chemical pesticides in the control of agricultural insect pests. (Ignoffo,
1973 and 1975, Falcon 1976 and 1977). This great impetus to use viral
pesticides is based on the environmental and ecological effects of toxic
chemical pesticides. For example, the three most commonly used chemical
insecticides, methylparathion, malathion and carbaryl, are highly toxic and
teratogenic to mammals. Several chemical insecticides, such as DDT, have
extremely high stability in nature. The accumulation of residual chemicals,
together with notable stability pose a great problem on environmental health.
The nuclear poly hydro sis viruses (NFVs) are known as a group of viruses
pathogenic to invertebrates. This group of viruses causes lethal disease in
their insect hosts. The virus particles of this group usually consist of
enveloped nncleocapsids which frequently are included in a large protein
lattice or polyhedron. The nucleocapsids are rod-shaped with dimensions of
about 250 x 50 mfi, and are usually singly enveloped; but in some instances,
more than one nucleocapsid can occur within one virus envelope. Viral genomes
of th s group were found to contain covalently closed supercoiled
double-stranded DNA with a molecular weight of approximately 75 to 100 x 10*
daltons (Summers and Anderson, 1973; Summers, 1977). These viruses comprise
the best known insect viruses. All together, the NFVs have been found in more
than 200 species of Lepidoptera, in 20 species of Hymenoptera and in 9 species
of Diptera (David, 1975). They are all in the genus Baculovirus.
These bacilliform viruses replicate in the nuclei of the infected cells.
During the process of infection, a substantial portion of virions is enveloped
and subsequently occluded in the protein matrix of polyhedra. The intact
polyhedra are not infectious in in vitro insect cell cultures, but they
are the key ''vector'' by which virus infections are transmitted in nature.
When insect larvae ingest the polyhedra, the infectious virions are released
from polyhedra through the solubilization of the protein matrix of polyhedra
in the alkaline environment and by enzymatic digestion in larvae gut (Harrap,
1972 and 1973). The purified NPV DNA was proved to be infectious in the
insect cell cultures (Ignoffo et al., 1971).
Several Baculoviruses have been used as viral pesticides for pest control
during the last 3 decades, e.g., the NPV of the Alfalfa caterpillar (Colias
eury theme) ; the NPV of cabbage looper (Trichoplasia ni); the NPV of
the beet army worm (Spodoptera exigua) and NPVs isolated from sawflies for
forest protection in the USA and Canada. T. ni was introduced to
Columbia, South America from California and has been used with great success
in recent years (Falcon, 1977).
Four NPV viral insecticides have been registered in the USA, and are
commercially available for field application. The first, ''Elcar'',
containing the NPV of the boll worm (Heliothis zea) is registered by the
pharmaceutical firm Sandoz, Inc., for the control of cotton bollworm. The
second available product named ' ' TM Bioctrol lr> is registered by the US
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Forest Service, and contains the NFV of the Douglas fir tussock moth (Orgvia
pseudot sugata). The NPV of the Gypsy Moth (Porthetria dispar) and NPV
of pine sawfly (Neodiprion sertifer) are two other NPVs which have been
registered.
With the hazardous environmental deterioration by chemical pesticides,
and with urgent need for promoting the world food production in mind, the use
of viral pesticides might be conceptually a practical and useful approach.
But before any great revolutionary events happen, a precise evaluation of the
benefit as well as the potential environmental health problem exhibited by
this approach should be made. It is estimated that in the western hemisphere,
30% of the current pest problems in agricultural crop production can be
treated with viral pesticides (Falcon, 1977). In California among the pest
species group causing major crop losses, 46% are susceptible to baculoviruses.
(Martignoni, 1975). Theoretically, viral pesticides can effectively solve
certain problems such as toxic chemical polution and inefficiencies of certain
chemical pesticides in crop production. As far as safety and environmental
health is concerned, relative amounts of in vivo and in vitro tests
have been performed. But most of the tests applied used acute infactivity,
antigenicity and morphological alteration as measuring factors. The fate of
viral DNA, possibilities of genetic recombination and viral gene integration,
viral oncogenicity as well as low level of persistent infection have never
been extensively examined.
As mentioned by Tinsley and Mel nick (1974), there are several important
considerations and noteworthy facts to be carefully examined and evaluated.
First, the candidate pesticidal virus may infect insect hosts other than the
target pest. Second, insect virus may be able to induce infection in other
invertebrate or even vertebrate via either permissive or abortive infection.
Third, as the consequence of persistent infection or non-fetal infection, the
insects are known to be carriers of a variety of animal aborviruses.
Pesticidal virus might follow the same pattern, and introduce itself into
human beings or other vertebrate through its vector host by an unnatural
route. Fourth, the so-called host specificity in virology is neither a
fundamental nor a stable characteristic; the condition of the host and the
nature of infectious agent (intact virion or naked DNA) will affect the entire
susceptibility to infection. Although numerous in vitro and in vivo
experiments have been done to prove the species specificity and the safety of
pesticide virus, the striking report of transfaction of Fogh-Lund human amnion
cell with the silk worm NPV-DNA (Himeno et al., 1967) and the demonstration of
viral DNA and antigens in vertebrate cells (Mclntosh and Eimura, 1974;
Granados 1976) have raised the question of species specificity and real
meaning of safety as monitored solely by the infectivity and cytopathic
effect. Furthermore, various cocarcinogens and tumor promoting agents, such
as phorbol ester, which probably exist widely in nature, might induce an
unexpected virus and host interaction which might lead to the onco genie
transformation of cells infected by pesticidal viruses.
In the application of pesticidal viruses, two important issues require
immediate attention. First of all, it is essential to improve the methodology
and sensitivity in detecting virus and host cell (including vertebrate cell
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and human cell) interaction at the molecular level and effects of cocarcinogen
on virus and host cell interaction; the alternative way of virus infection,
the fate of viral DNA, possible viral gene integration and recombination,
viral oncogenicity and persistent infection require a molecular biological
method of detection and observation other than infectivity assay. Secondly,
the structure, function and genetic relatedness of baculovirus have to be
carefully studied and examined; a universal pesticidal virus or a
multifunctional pesticidal virus may be constructed.
Other than the classic methods of detection and analysis, there are
several recent major technical approaches which can be applied to insect virus
systems and will add a great impact to the understanding of viral genome
status, gene structure, gene function and pathogenesis. Such as:
(a) Nucleic acid hybridization (including DNA-DNA reas sociation
kinetics analysis, _iji _§_i_tji RNA—DNA cy t ohybr idiz a ti on.
Southern's blot hybridization, etc.)
Detection of viral DNA, defective or non-defective, can be achieved
by DNA-DNA reassociation kinetics analysis (Huang and Pagano, 1977). Using
highly specific radioactive viral DNA probes, it has been possible to detect
small numbers of copies or portions of viral genomes in the DNA isolated from
cells suspected of carrying viral information. It does not matter whether
viral DNA is replicating or defective, integrated or pi asm id, biologically
active or latent. This technique is able to tell the degree of homology and
relatedness between two viruses or two individuals. The degree of viral gene
expression, in regard to tr an script ional mRNA, can also be detected by this
technique.
As far as localization of viral nucleic acid and detection of
susceptible cell types is concerned, the technique of in situ RNA-DNA
cytohybridization will fulfill the goal (Huang et al., 1973, Huang and Pagano,
1977). The great advantage of this technique is. its ability to localize
virus-specific DNA or RNA according to cell type and intracellular location by
aut or adi ogr aphy. In combination with these nucleic acid hybridization
techniques, a more advanced study of the interaction of insecticidal virus
with the mammalian cell, especially human cells, can be achieved.
(b) Restriction endonuclease and specific DNA fragmentation.
The DNA fragmentation by restriction endonuclease has become a very
powerful tool for analyzing not only small viral genomes but also genomes of
increasing complexity and molecular size. Cleavage of DNA into specific
terminal fragments and construction of a DNA fragment map will provide
elements needed for the detailed characterization of viral genome, and also
for the regulation of gene transcription and gene interaction. The
restriction enzyme cleavage pattern will also provide a detailed comparison of
strain variation and strain relatedness.
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In a de no vims system, by DNA fragment transfection and DNA-DNA
reassociation kinetics analysis (usiing restriction endonuclease fragments as
probe), it was found that only the extreme left-hand 7% of the adenovirus type
2 DNA is sufficient for transformation of rat kidney cell in vitro
Gallimore 1974; Graham et al., 1974). The EcoHl-C fragment, the left 16% of
the viral genome, of adenovirus type 12 DNA has been proved to carry a
transforming gene, and was used as a powerful probe for the study of the
association of adenovirus type 2 with various types of human cancer (Hackey et
al., 1976).
The structure and function of several viral genomes such as
0X174, SV40, adenovirns, etc., have been elucidated by the application of
restriction endo nucl ease s . Using DNA fragments generated by various
restriction enzymes and nucleic acid hybridization techniques, the virus gene
expression and gene regulation in SV40 and adenovirns-infected permissive and
non-permissive cells have been defined. The utilization of restriction
endonuclease and nucleic acid hybridization in the human cy tome gal ovirus
system has been very successfully performed in our laboratory. We feel that
these techniques can be effectively applied to study gene interaction and gene
expression in pesticidal virus-infected permissive and non-permissive cells.
(c) Transfection of viral DNA using calcium phosphate and
dime thy Isulf oxide (DMSO).
Viral infection can be initiated in an alternate route in an in
vitro system. By infection of cells treated with calcium phosphate and
DHSO, adenovirus DNA and herpes simplex DNA have been proved to be infectious
(Graham and Van Der Eb 1973a). It is not necessary to have intact virus
particles to initiate the infection process. Transformation of rat cells by
DNA of adenovirus type 5 was also achieved by this method (Graham and Van Der
Eb, 1973b). As mentioned above, the specific DNA fragment carrying the
transforming gene has also been detected by calcium phosphate method. Using
this technique to advantage, there is an urgent need for the examination of
the biological activity of pesticidal viral DNA. Mass application of
pesticidal virus will generate numerous defective or naked DNA and on some
occasions these particles might become a potential environmental hazard and
dangerous to human health.
(d) Gene cloning and recombinant DNA technology.
Gene cloning and recombinant DNA technology has become a
revolutionary tool not only for the study of molecular biology but also for
industrial application. Numerous genes of biochemical and genetic interest
have been isolated and studied due to the achievements of recombinant DNA
research. Virus genomes can be constructed and amplified in vitro without
the natural hosts, and a wide host range, non-hazardous pesticidal virus might
therefore be constructed with a minimum risk to health and environment.
This report contains three main elements which reflect the work we
have performed with the support of a grant from EPA: the interaction of SfNPV
with various mammalian cells in vitro, the genomic structure of SfNPV and
cloning of SfNPV DNA (Hind III fragments) in plasmid pBR322. The details are
described in the following sections.
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REFERENCES
1. Falcon, L.A. 1978. Viruses as alternatives to chemical pesticides in
the western hemisphere. In Viral Pesticides; Present Knowledge and
EPA-600/9-7 8-026. Summers, M.D. and Kawanishi, C.T. , eds.
2. Falcon, L.A. 1976. Annual Rev. Entomology 21: 305-324.
3. David, W.A.L. 1975. Ann. Rev. Entomol. 20: 97-117.
4. Gallimore, P.H. 1974. J. Gen. Virol. 25: 26.
5. Graham, F.L. and Van Der Eb, A.J. 1973a. Virology 52: 456-467.
6. Graham, F.L. , and Van Der Eb, A.J. 1973b. Virology 54: 536-539.
7. Granados, R.R. 1976. Adv. Virus Research 20: 189-236.
8. Harrap, K.A. 1972. Virology 50: 114-123.
9. Harrap, K.A. 1973. Virus infection in invertebrates, pp. 271-299.
In: A.J. Gibbs (ed. ) Virus and Invertebrates. North Holland Publishing
Co., Amsterdam.
10. Himeno, M.F., et al. 1967. Virology 30: 507-512.
11. Huang, E. S. , Chen, S.T. , and Pagano, J.S. 1973. J. Virology 12:
1473-1481.
12. Huang, E. S. , Newbold, I.E. and Pagano, J.S. 1973. J. Virology 11:
508-514.
13. Huang, E. S. , and Pagano, I.S. 1977. Nucleic Acid Hybr idizatioion
Technology and Detection of Proviral Genome. In Methods in Virology,
Vol. 6, pp. 457-497 (ed. Haramorosch and Koprowski) . Academic Press,
New York.
14. Ignoffo, C.M. 1973. Ann. New York Acad. Sci. 217: 141-172.
15. Ignoffo, C.M. 1975.. Environ. Letters 8: 23-40.
16. Ignoffo, C.M., Shapiro, M. , and Hink, W.F. 1971. J. Invertebrate
Pathol. 18: 131-134.
17. Macky, I.E., and Rigden, P.M., et al. Proc. Natl. Acad. USA 72: 4657.
18. Mclntosh, A., and Kimura, M. 1974. Intervirology 4: 257-267.
19. Summers, M. D. 1977. Deozyribonucl ei c Acids of Baculoviruses. In
''Virology in Agriculture''. pp. 233-246. John A. Romberger.
All ante Id, Osmun and Co. Publishers, Inc.
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20. Summers, M.D. and Anderson, D.L. 1973. J. Virol. 12: 1336-1346.
21. Tinsley, T.W. and D.J.L. Mel nick. 1973. Intervirology 2: 206-208.
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II. CONCLUSIONS AND RECOMMENDATIONS
Spodoptera frugjperda nuclear polyhydrosis virus DNA has been
purified from polyhedra of the Ohio, North Carolina and Georgia strains.
Viral DNA has been characterized for its size, electron microscopic morphology
and base distribution. Viral genomes have been further studied by restriction
fragmentation for genomic structure and srain variation. Viruses isolated
from different geographical areas have their own identities in DNA restriction
patterns.
To facilitate the molecular biological study of this virus, a DNA
restriction fragment map has been made, and a library of recombinant clones
carrying inserts of various viral DNA fragments have been constructed. The
availability of DNA fragment map and the library of recombinant clones will
make further molecular biological studies of SfNPV more fruitful.
Virus from h emolymph of SfNPV infected insects and viral DNA purified
from polyhedra have been used to infect and transfect mammalian cells and
insect cells from its host.
No productive infection or morphological transformation have been
observed in mammalian systems, while positive infection was observed in the
cell from its own host. The application of advanced nucleic acid
hybridization confirmed the immunological as well as infectivity results.
Based on these observations it is concluded that SfNPV does not cause severe
harm to human, murine or other rodent cells in vitro. The extension of
these studies to other insect viruses and to other mammalian cell systems is
needed before there is extensive application of pesticidal viruses in the
field.
Recombinant DNA research is well advanced in the field of biology in this
country. It is essential for government to encourage scientists to devote
time and effort on the construction of multi-variant pesticidal virus with no
pathogenicity to humans and other mammals. The success in this field may
alleviate the problem of toxicity created by chemical pesticides.
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II. SCIENTIFIC EXPERIMENTS AND RESULTS
Section A. General Methods for Purification of S. frugiperda NPV and Viral
DNA
Virus Purification. The partial purification of polyhedra of the Ohio
strain of SfNPV was performed at the Southern Grain Insects Laboratory from
the lysate of virus— infe ct ed Fall army worm larvae in Tifton, Ga.
Subsequently, this suspension of polyhedra was washed by repeated
centr if ugation in a Sorvall RC-S refrigerated centrifuge at 5,000 rpm for 5
min and resuspended in distilled water until the supernatant fluid was clear.
After the last centrifugation, the polyhedra were suspended in a freshly
prepared solution of 0.1M Na2c03 for 15 to 20 min at room temperature and
centrifuged for 5 min at 5,000 rpm. This procedure was repeated two to three
times until the alkaline dissolution of the polyhedra was completed. The
supernatant from each centrifugation was pooled, layered gently onto 20 to 60%
(wt/wt) sucrose gradients made in TBS (0.15 M NaCl-0.05 M
Tri s-hydro chl or ide, pH 7.4) and centr if uged in an SW-27 rotor for 75 min at
25,000 rpm and 4°C. The multiple virus bands in each gradient were
collected through the bottom of each tube, dialyzed against TBS overnight, and
stored at 4°C. If a more concentrated virus preparation is desired, the
virions may be pelleted by centr if ugation and resuspended in an appropriate
buffer.
Ejc_t .r.a_c.t.i.o.n_.o_f _ v_i.r.a.l_.DN A . A concentrated suspension of
gradient-purified virions was digested at 55°C for 1.5h in a proteinase K
digestion mixture (1% sodium dodecyl sulfate-5 mM CaCl2-l mM EDTA-100 |ig of
proteinase E per ml). When pronase (1 rag/ml) was substituted for proteinase
K, the digestion was carried out at 37°C.
The digested mixture was carefully layered onto preformed 10 to 30%
(wt/vol) sucrose gradients in TBS containing 1 mM EDTA and centrifuged in
SW-27 rotors at 18,000 rpm for 18h in a Beckman L3-40 nl tr acentr if uge.
Fractions (1 ml) were collected through the top by pumping a 60% sucrose
solution through the bottom of the tube. The DNA-containing fractions as
determined by TTV absorption were pooled, dialyzed against 0.05 M
Tris-hydroxychloride (pH 7.4)-l mM EDTA buffer overnight and stored at 4°C.
Alternatively, a modification of the method of Radloff et al. (1967) was
used. The proteinase E-digested mixture was diluted with 0.01M
Tris-hydrochloride (pH 8.0)-0.01 M EDTA buffer containing ethidium bromide at
a concentration of 200 ng/ml. Cesium chloride was added to the solution
until the density was 1.59 ng/ml. Centrif ugation was carried out in a VI-50
rotor at 36,000 rpm for 24 h in a Beckman ul tr acentr if uge. The DNA-containing
fractions were visualized by their fluorescence in UV light and collected.
The ethidium bromide was removed by extraction with isoamyl alcohol, and the
DNA fractions were dialyzed against 0.05 H Tris-hydrochloride (pH 7.4)-0.001M
EDTA buffer overnight and stored at 4°C or further purified by sedimentation
through a sucrose gradient as previously described.
If intact DNA molecules were not required, the DNA solution could be
extracted with phenol, and the DNA could be precipitated with cold ethanol at
-20°C and redissolved in an appropriate buffer.
-------
REFERENCES
Radloff, R. , W. Bauer and J. Vinogard. 1967. A dye-buoyant density
method for the detection and isolation of closed circular duplex DNA:
The closed circular MA in HeLa cells. Proc. Natl. Acad. Sci. USA 57:
1514-1521.
-------
Section B; Analysis of the Spodoptera frugiperda
Nuclear Polyhedrosis Virus Genome by Restriction
Endonucleases and Electron Microscopy
(published in J. of Virology, Vol. 44, 747-751, 1982)
Lambert C. Loh1, John J. Hamm2, Clinton Kawanishi* and
Eng-Shang Huang1
1 Cancer Research Center, University of North Carolina, Chapel Hill, North
Carolina 27514. * Southern Grain Insects Laboratory, U.S. Department of
Agriculture, Tifton, Georgia 31793. 'U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina 27711.
ABSTRACT
Restriction endonuclease analysis was used to differentiate between four
strains of Soodoptera frugiperda nuclear polyhedrosis virus from different
geographical areas. In addition, partial denaturation was performed, and a
partial denaturation map was constructed for the Ohio strain of this virus.
EXPERIMENTAL METHODS AND RESULTS
With the increasing interest in the use of insect viruses as agents for
the biological control of insect pests, there is an urgent need to identify
and characterize insect viruses and various virus isolates. In this report,
the restriction endonnclease patterns of DNA from a strain of Spodoptera
frugiperda nuclear polyhedrosis virus (SfNPV) for BamHI, EcoRI, and Hindlll
were determined and used to differentiate between. SfNPV isolates from Georgia
(GA), Mississippi (MS), North Carolina (NC), and Ohio (OH). In addition, a
partial denaturation map of the OH strain of SfNPV was constructed.
The strains of SfNPV were originally isolated from diseased fall army-worm
larvae at Tifton, Ga. ; Starkville, Miss.; Plymouth, N.C.; and Cleveland,
Ohio. The virions were purified from the lysate of virus-infected fall
armyworm larvae by differential centrifugation and sucrose gradients as
previously described (8). The extraction of DNA from the virions, its
digestion by restriction endonuclease s, and the in vitro labeling of DNA
restriction fragments and their visualization after agarose gel
electrophoresis were performed essentially as described in our previous report
(8).
The partial denaturation map was constructed as follows. Purified viral
DNA was partially denatured by a modification of the method of Inman and
Schnos (3) as described by Wadsworth et al. (11) and Kil pa trick and Huang (5).
Specifically, a 10 fil sample of DNA (6 to 10 ng/ml) was mixed with an equal
volume of denaturation buffer at room temperature and allowed to react for 7
min. The denaturation buffer consisted of 20% (vol/vol) formaldehyde, 0.02
M Na2C03. 5 mM EDTA, and enough NaOH to bring the pH up to an appropriate
value. It was found empirically that a pH of 11.15 gave the most distinct
partial denaturation pattern, and denaturation was already quite extensive at
10
-------
pH 11.25. The reaction was stopped by the addition of 80 p.1 of ice-cold
spreading solution consisting of 70 |il of 1M ammonium acetate, 5 fil of 0.2H
acetic acid, and 5 |il of cytochrome c (2 mg/ml) per 20 |il of the denatured
DNA solution. The pH of the final solution was about 5.2.
The aqueous method (6) of spreading partially denatured DNA molecules
(5,11) was used to prepare the specimen grids. Immediately after the
termination of partial denaturation, 1 fil each of denatured and completely
alkaline-denatured 0X174 RF molecules were added to the reaction mixture
as internal length standards. A 50 (il amount of this solution was spread over
the surface of an 0.3 H ammonium acetate solution adjusted to pH 5.2. The
DNA-cy t ochrome c film was immediately transferred to parl odion-coa ted,
200-mesh copper grids by surface contact, stained with uranyl acetate,
dehydrated in 90% ethanol, rotary shadowed with platinum-palladium (80:20)
alloy, and stabilized with a carbon coating to minimize distortions from the
electron beam.
The sample grids were examined in a Hitachi H—500 electron microscope at
50 kV. The electron micrographs of DNA molecules were taken at magnifications
ranging from 3,000 to 9,000. The micrographs were enlarged by an overhead
projector, and only intact, circular, relatively untangled DNA molecules were
used for length measurements. A programmed Hewlett-Packard 9825A calculator
and digitizer was used to trace the projected DNA molecules, and lengths were
recorded in microns. 0X174 RF DNA, with a known molecular weight of
3.48 x 106 (10), was used as a standard. For partially denatured molecules,
the lengths of the single-stranded and double-stranded regions were measured
separately. The lengths of the single-stranded regions were then corrected
for shrinkage by a factor of 1.418, a value obtained empirically by comparing
the molecular lengths of alkaline-denatured and intact 0X174 RF DNA
molecules cospread with the partially denatured SfNPV DNA molecules.
The OH strain of SfNPV was chosen for detailed analysis. The buoyant
density of the viral DNA was found to be 1.6992*0.0003 g/ml by equilibrium
CsCl gradient centrifugation in a Spinco model E analytical ultracentrifuge,
with Micrococcus lysode ikt icus DNA used as a density marker (p=1.731
g/ml). Thus, the viral DNA should have an average guanine plus cytosine
(G+C) content of 40% as calculated by the equation derived by Schildkraut et
al. (9). The molecular weight of the viral genome was found to be 8.25
(±5.2) x 10* by electron microscopy. By these parameters, it was
virtually indistinguishable from the genome of the GA strain of SfNPV, from
which the SfNPV strains propagated inmost other laboratories in the United
States and Europe were originally derived. The molecular weight and density
data for SfNPV DNA obtained in our laboratory agree reasonably well with the
values reported previously (1,2,4,7).
The SfNPV OH genome was cleaved into 8, 15, and 25 fragments by the
restriction endonucl ease s BamHI, Hindlll, and EcoRI, respectively (Fig. 1).
The molecular weights of these restriction fragments and their designations
were reported in a previous paper (8).
11
-------
Viral DNA from the GA, MS, OH, and NC strains SfNFV were cleaved with
BamHI, Hindlll, or EcoRI, end-labeled, and electrophoretically separated on
0.7% agarose gels. The resulting autoradiographs are shown in Fig. 2. The
migration patterns of the Hindlll digests were identical for the MS, NC, and
OH strains. The extra fragment present in the GA srain may be due to
heterogeneity within the virus preparation. The EcoRl digests of the GA and
MS strains had migration patterns that were easily distinguishable from those
of the NC and OH strains. Heterogeneity may account for the presence of some
of the submolar fragments observed. Loss of EcoRl sites, possibly between
some of the linked comigrating fragments such as EcoRl fragments C and D, may
also explain the appearance of extra high-molecular-weight restriction
fragments (e.g., the EcoRl fragment above EcoRl-A in the MS digest [Fig. 1C
and 21 ).
12
-------
50
100
150
200
C|
C2
50
100
150
200
50 IOO 150
MIGRATION DISTANCE
( mm )
200
Fig. i. Mi erode nsi t ome ter scans of ant oradiographs of electrophor eti cally
separated, end-labeled SfNFV OH DNA cleaved by (a) BamHI, (b) Hindlll, or (c)
EcoRl . Fragment S in the EcoRl digest is the only fragment present in
submolar (0.5 mol) amounts. All cleavage patterns were scanned at a 1:1
scan-to-record ratio.
13
-------
Virions usd for DNA purification were purified from the lysate of
virus-infected larvae cloned in vivo. In vitro plaque purification of
the various virus strains was not done because of the lack of a sound
permissive cell system which can generate infectious virus in SfNPV-infected
cell cultures. Therefore, confirmation of the loss of a specific restriction
enzyme site must await DNA sequencing data and the construction of a complete
restriction map of the viral genome of EcoRl. On the other hand, the
migration patterns of the BamHI digests were quite distinct for each of the
four strains of SfNPV. The fact that SfNPV OH had the largest number of BamHI
sites was one reason this strain was chosen for detailed analysis and
restriction mapping in our laboratory. From the known restriction map for
BamHI (8) and the sizes of the BamHI restriction fragments, we can deduce that
the NC strain might have lost the BamHI site between BamHI fragments A and
G; the MS strain has lost BamHII fragments A and D, and the GA strain has
lost the BamHI site between BamHI fragments A and D (Fig. 1 and 2). Again, a
final conclusion about the loss of BamHI sites in these cases can only be made
with the support of DNA sequencing data.
Preliminary experiments showed that the nick—translated BamHI-G and D/E
fragments eluted from gels of a BamHI digest of SfNPV OH DNA did hybridize to
the largest BamHI restriction fragment of the other strains, as predicted.
, Hind in aamHf EccR!
.NC OH M5 GA NC OH MS GA NC OH 'VtS GA
»«* *•»*"* ***
. «***«*»
Fig 2. Cleavage patterns of DNAs from the NC, OH, MS, and GA strains of
SfNPV with restriction endonucl e ase s BamHI, Hindlll, and EcoRI. The
restriction fragments were end-labeled with [a-JaP]dATP and
electrophoreti cally separated in an 0.7% agarose gel.
-------
s
c :
°rientin* the circular viral DMA molecule, we
,
11.25.
*
the pH w>i r.ised .boy. 11.25, .xt.i.iv.
'"'
p"tial
Bar,
the denatured single-stranded region.
15
Reproduced from
best available copy.
-------
We facilitated the data analysis by photographing only relaxed circular
DNA molecules for length measurements. Because of the slight length
variations between different DNA spreads, it was decided that the best way to
compare the data from different experiments was to express all single- and
double-stranded lengths as a percentage of total circular length instead of as
absolute length units. It was found that with few exceptions, after
correction for single-stranded shrinkage, the total circular lengths of
partially denatured molecules were comparable to those of undenatured DNA
molecules spread under similar conditions as controls. This justified the use
of the shrinkage factor of 1.418 described above.
During the alignment of the partially denatured molecules,
representations of these DNA molecules were plotted on strips of graph paper
on a scale of 10% of the total length per inch. The molecules were then
arranged for maximum overlap between the few A+T-rich and G+C-rich regions.
The frequency of occurrence of the denaturation sites along the DNA of 14
molecules examined was then calculated to give the tentative partial
denaturation map (Fig. 4).
Jin
LAA
0 10 20 30 40 50 SO 70 80 90 100
% TOTAL LENGTH
0 IO 20 3O "JO 50 60 70 80 90 100
% TOT4L LENGTH
4. Histograms showing the positions and frequencies of denatured sites
for SfNPV OH DNA after partial denaturation at (a) pH 1.15 or (b) pH 11.25.
The Y axis represents the number of denatured sites per 2% of the total length
(a) or the number of denatured sites per 1% of the total length (b).
Beginning from the origin of the map (Fig. 4a), there was a major
relative A+T-rich zone which extended for about 15% of the total length.
There followed a region of lower A+T content that stretched for about 30% of
the molecule; at pH 11.25 (Fig. 4b) most of this region was denatured, but
at 11.15 there were a few small, relatively G+C-rich sites scattered around
this region. The first G+C zone was found to be located immediately next to
the central portion of the map and could only be recognized at pH 11.25.
Another G+C-rich zone spanned the terminal 15% of the map. These two regions
were the only ones that remained undenatured at pH 11.25. The region between
them was marked by two relative A+T-rich sites.
16
-------
In summary, we characterized four geographically different strains of
SfNPV by restriction endonuclease digestion. In addition, a partial
denaturation map of the SfNPV OH genome was constructed. There was no
indication of the presence of long stretches of high G+C or high A+T regions
or of highly repetitive genome sequences, as was the case with certain
herpesviruses. However, the asymmetrical pattern of the two G+C-rich regions
shown in the denaturation map at pH 11.25 might provide a means for orienting
the circular viral DNA molecule.
REFERENCES
1. Bud, H.M. , and D. C. Kelly. 1977. The DNA contained by nuclear
polyhedrosis viruses isolated from four Spodoptera spp. (Lepidoptera,
Noctuidae): genome size and configuration assessed by electron
microscopy. J. Gen. Virol. 37: 135-143.
2. Harrap, K.A., C. C. Payne, and I.S. Robertson. 1977. The properties of
three baculbviruses from closely related hosts. Virology 79: 14-31.
3. Inman, R.B., and M. Schnos. 1970. Partial denaturation of thy mine- and
5-bromouracil-containi ng •> DNA in alkali. J. Mol. Biol. 49: 93-98.
4. Kelly, D. C. 1977. The DNA contained by nuclear polyhedrosis viruses
isolated from four Spodoptera Sp. (Lepidoptera, Noctuidae): genome size
and homology assessed by DNA reas sociation kinetics. Virology 76:
468-471.
5. Kilpatrick, B.A., and E.-S. Huang. 1977. Hunan cytomegalovirus genome:
Partial denaturation map and organization of genome sequences. J.
Virol. 24: 261-276.
6. Kleinschmidt, A. K. 1968. Honolayer techniques in electron microscopy
of nucleic acid molecules. Methods Enzymol. 22: 361-377.
7. Knudson, D.L., and T.W. Tinsley.. 1978. Replication of a nuclear
polyhedrosis virus in a continuous cell line of Spodoptera
f rugiperda; Partial characterization of viral DNA, comparative
DNA-DNA hybridization and pattern of DNA synthesis. Virology 87: 42-57.
8. Loh, L. C., J.J. Hamm, and E.-S. Huang. 1981. Spodoptera fruniperda
nuclear polyhedrosis virus genome: Physical maps for restriction
endonucleases BamHI and Hindlll. J. Virol. 38: 922-931.
9. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Determination of the
base composition of deoxyribonucleic acid from its buoyant density in
CsCl. J. Mol. Biol. 4: 430-443.
10. Sharp, P. A., M. T. Hus, E. Ohtsubo, and N. Davidson. 1972. Electron
microscope heteroduplez studies of sequence relations among pi asmids of
E. col i. I. Structure of F-prime factors. J. Mol. Biol. 71:
471-497.
17
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11. Wads worth, S. , R. J. Jacob, and B. Roizman. 1975. Anatomy of herpes
simplex virus DNA. II. Size, composition, and arrangement of inverted
terminal repetitions. J. Virol. 15: 1487-1497.
18
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Section C; Spodoptera frugjperda Nuclear
Poly hedrosis Virus Genome: Physical Naps for
Restriction Endonucleases BamHI and Hindlll
(published in J. of Virology. Vol. 38: 922-931, 1981)
Lambert C. Loh1, John J. Hamm2, and Eng-Shang Huang1
1Cancer Research Center, University of North Carolina, Chapel Hill, North
Carolina 27514. *U.S. Department of Agriculture, Southeast Area, Southern
Grain Insects Research Laboratory, Tifton, Georgia 31793.
ABSTRACT
The physical map for the genome of Spodoptera frugiperda nuclear
polyhedrosis virus was constructed for restriction endonucleases BamHI and
Hindlll. The ordering of the restriction fragments was accomplished by
cross-blot hybridization of BamHI, Hindlll, and EcoRl fragments. The
alignment of the Hindlll fragments within the BamHI map was achieved by double
digestion with the two restriction endonucleases followed by cross—blot
hybridization. The results showed that the viral genome consisted of mainly
unique sequences. In addition, the circular nature of the viral genome was
reaffirmed.
INTRODUCTION
Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV) is a member
of the baculovirus group with a double-stranded, circular, supercoiled DNA
genome (16). The molecular weight of the DNA molecule has been determined to
be about 80 x 10* by electron microscopy and restriction endonuclease
analysis in this laboratory (J. Virol. 44: 747-751, 1982), in reasonable
agreement with previous reports by other investigators (1,2,4,7,8,16).
Because of its relatively large size and complexity, the construction of
a restriction map of the viral genome should greatly facilitate the study of
its molecular biology. It would allow investigators to study virus
recombinants and map the crossover sites (14,17). The comparison of different
virus strains by restriction endonuclease analysis (9,13) is more meaningful
when it is possible to locate the regions showing the greatest genetic
variation between virus strains. Even more important, it is essential for the
study of gene transcription and regulation in both permissive and
non-permissive cells. In this paper, a physical map of the SfNPV genome was
constructed with the restriction endonucleases BamHI and Hindlll.
MATERIALS AND METHODS
Virus purification. The partial purification of polyhedra of tthe Ohio
strain of SfNPV was performed at the Southern Grain Insects Laboratory from
the lysate of virus-infe ct ed Fall army worm larvae in Tifton, Ga.
Subsequently, this suspension of polyhedra was washed by repeated
19
-------
centrifngation in a Sorvall RC-S refrigerated centrifuge at 5,000 rpm for 5
min and resuspension in distilled water until the supernatant fluid was clear.
After the last centrif ugationn, the polyhedra were suspended in a freshly
prepared solution of 0.1 M Na2C03 for 15 to 20 min at room temperature and
centr if uged foor 5 min at 5,000 rpm. This procedure was repeated two to three
times until the alkaline dissolution of the polyhedra was complete. The
supernatant from each centrifugation was pooled, layered gently onto 20 to 60%
(wt/wt) sucrose gradients made in TBS (0.15 M NaCl-0.05M Tris-hydrochloride,
pH 7.4) and centrif uged in a SW-27 rotor for 75 min at 25.000 rpm and 4°C.
The multiple virus bands in each gradient were collected through the bottom of
each tube, dialyzed against TBS overnight, and stored at 4°C. If a more
concentrated virus preparation is desired, the virions may be pelleted by
centrifugation and resuspended in an appropriate buffer.
.E.5.t.r.a.c.t .i.o.n_.o.f _.Y_i.r.a.l_.DlLA • A concentrated suspension of
gradient-purified virions was digested at 55 °C for 1.5 h in a proteinase K
digestion mixture (1% sodium dodecyl sulfate-5 mil CaCl2-l mH EDTA-100 ng of
proteinase E per ml). When pronase (1 mg/ml) was substituted for proteinase
K, the digestion was carried out at 37 °C.
The digested mixture was carefully layered onto preformed 10 to 30%
(wt/vol) sucrose gradients in TBS containing 1 mH EDTA and centrifuged in
SW-27 rotors at 18,000 rpm for 18 h in a Beckman L3-40 ultracentrifuge.
Fractions (1 ml) were collected through the top by pumping a 60% sucrose
solution through the bottom of the tube. The DNA-containing fractions as
determined by TJV absorption were pooled, dialyzed against 0.05 M
Tris-hydrochlor ide (pH 7.4)-l mM EDTA buffer overnight and stored at 4°C.
Alternatively, a modification of the method of Radloff et al. (10) was
used. The proteinase K-digested mixture was diluted with 0.01 M
Tris-hydrochloride (pH 810)-0.01 M EDTA buffer containing ethidium bromide at
a concentration of 200 ug/ml. Cesium chloride was added to the solution
until the density was 1.59 g/ml. Centrif ugation was carried out in a VI-50
rotor at 36,000 rpm for 24 h in a Beckman ul tracentr if uge. The DNA-containing
fractions were visualized by their fluorescence in UV light and collected.
The ethidium bromide was removed by extraction with isoamyl alcohol, and the
DNA fractions were dialyzed against 0.05 M Tris-hydrochloride (pH 7.4)-0.001 M
EDTA buffer overnight and stored at 4°C or further purified by sedimentation
through a sucrose gradient as previously described.
If intact DNA molecules were not required, the DNA solution could be
extracted with phenol, and the DNA could be precipitated with cold ethanol at
-20°C and redissolved in an appropriate buffer.
Restriction enzyme digestion of viral DNA. Purified viral DNA was
digested with EcoRl, BamHI, or Hindlll in a solution containing 0.05 M
Tris-hydrochloride (pH 7.4)-0.01 M MgCl2~0.10 M NaCl-0.006 M
p-mercapt oe th anol. Incubations were generally for 3 h at 37°C, and
sufficient enzyme was added for complete digestion within this period. When
partial digestion of the viral DNA became necessary for mapping purposes, the
same amount of restriction enzyme was added, but incubation was carried out at
20
-------
10°C for between 10 and 30 min. The reaction was stopped by adding EDTA to
a final concentration of 10 mM. The restriction enzyme was then heat
inactivated at 70°C for 20 min.
The restriction enzyme EcoRl was purified from Escherichia coli
strain Ryl3 (3), Hindlll was purchased from Bethesda Research Laboratories,
Inc., Rockville, Md., and Bam HI was purified from fi.a.c_i.l_l.u_s
amvloliquefaciens H (19; L.A. Smith and J.G. Chirikjian, Fed. Proc. 36:
908. 1977).
In vitro labeling of DNA. For critical determinations of the molecular
weights and stoichiometry of DNA fragments by electrophoresis in agarose gels,
the DNA restriction fragments were end labeled iji vitro with avian
myel obi astosi s virus reverse tr anscriptase obtained from G.E. Houts of the
Life Sciences Institute, St. Petersburg, Fla. Approximately 10 uCi of
[o-3aP]dATP (450 Ci/mmol; ICN Pharmaceuticals) were lyophilized in a
small tube and redissolved in a reaction mixture containing 20 to 25 (ig of DNA
restriction fragments per ml, 100 mM and NaCl, 50 mM Tris-hydrochloride (pH
7.4), 6 mM B-mercaptoethanol, 10 mM MgCl2, 1 mM MnCl2, 0.1 mM dCTP, 0.1
mM dGTP, and 0.1 mM dTTP. Reverse tr anscripta se (15 D) was added, and
incubation was carried out at 4°C for 20 to 30 min. The reaction was
stopped by heat inactivating the enzyme in a 70°C water bath for 20 min.
The mixture was then treated with proteinase K (50 ug/ml) for 30 min at 37°C
before gel electrophoresis.
For cross-blot hybridization experiments, labeling was done with DNA
polymerase I instead of reverse tr anscriptase. The procedure, a modification
of the method of Rigby et al. (12), was essentially the same as that described
in the preceding paragraph, except that 5 ul of DNA polymerase I was used, the
MnCl2 was not needed, and incubation was carried out at 14°C for 30 min.
It was found that this procedure can be adopted for end labeling DNA
restriction fragments as well. The DNA polymerase I used in these experiments
was purified by the method of Richardson et al. (11) as modified by Jovin et
al. (6).
Agarose gel electrophoresis of DNA fragments. The DNA restriction
fragments were fractionated by electrophoresis through 0.7% agarose (SeaKem)
gels. Agarose was dissolved in E buffer (0.04 M Tris-hydrochloride-0.02 M
sodium acetate-1 mM EDTA, pH 7.2) and allowed to solidify on a glass plate to
form a horizontal slab gel (6 by 200 by 250 mm). For cross-blot hybridization
experiments, slightly wider gels (6 by 250 by 250 mm) were used. Sample slots
of suitable sizes were formed by inserting a plexiglas comb at one end of the
gel before gel formation was complete. DNA samples were loaded into the slots
with tracer amounts of a bromophenol blue solution made up in E buffer with
30% glycerol.
The DNA was el ectr ophor esed at a constant voltage of 100 V at 25°C for
about 18 h until the bromophenol blue marker reached the end of the gel.
Visualization of DNA fragments. When unlabeled DNA samples were
el ectr ophor esed, the gel was stained with ethidium bromide (20 ug/ml in E
buffer) for Ihr, and the DNA fragments could be visualized under UV light and
photographed with a Polaroid MP-4 camera with an orange filter under the lens.
21
-------
In the case of labeled DNA samples, the gel was dehydrated by vacuum on a
gel dryer and exposed to Kodak X-Omat R X-ray film for 24 to 48 h. The
positions of the DNA fragments were thus recorded on the developed film. For
molecular weight measurements, lambda DNA digested with EcoRl, BamHI, or
Hindlll was used for molecular weight markers. The migration distances of the
DNA fragments as recorded on film were determined by a Hewlett Packard 9825A
calculator and digitizer programmed to calculate the DNA fragment sizes in
kilobases when suitable molecular weight markers were provided. Densitometer
tracings of the autoradiographs were done with a Joyce Loebl
microdensitometer. The molar ratios of the DNA fragments, being proportional
to the areas under the peaks, were then determined by cutting out and weighing
the corresponding peaks on these tracings.
Cross-blot hybridization. To detect homology between different
fragments, the Hutchison cross-blot hybridization technique (C. Hutchison,
personal communication) was used. A ''cold'' restriction digest was run
across the entire width of a square slab gel (6 by 250 by 250 mm) using 10 to
15 ug of viral DNA per gel. The positions of the DNA bands could be
photographed under UV light after staining with ethidium bromide. The DNA in
the gel was then depurinated partially with 0.25 H NaOH-1 M NaCl, and
neutralized in 1 M Tri s- hydro chloride (pH 7.4)-1.5 M NaCl to improve the
efficiency of transfer of large DNA fragments onto a nitrocellulose sheet
(18). The actual transfer was done in 6 x SSC (1 x SSC is 0.15 M NaCl plus
0.015 H sodium citrate) at room temperature for 8 h by the procedure of
Southern (15) with slight modifications. The nitrocellulose sheet (Bethesda
Research Laboratories) was then air dried briefly and baked in vacuum at
80°C for 3 h.
A **P-labeled restriction digest was run and prepared for transfer as
described above. The nitrocellulose sheet with the immobilized DNA wsa placed
on top of the radioactive gel such that each cold band bound to the
nitrocellulose sheet intersected each ''hot'' band in the gel at right angles.
The hot band patterns were then transferred and directly hybridized at right
angles to the unlabeled DNA bands on the nitrocellulose sheet in hybridization
buffer (6 x SSC-0.1% sodium dodecyl sulfate) at 65°C before the transfer and
hybridization step. Finally, the nitrocellulose sheet was washed as described
by Jeffreys and Flavell (5), blotted dry, wrapped in a protective plastic
freezer bag, and exposed to Kodak 0-Omat R X-ray film for 3 days. A DuPont
Lightning-Plus intensifier screen was often used to shorten the exposure time.
After development, spots of developed silver grains should mark the
intersection points of DNA fragments sharing common sequences.
RESULTS
Restriction enzyme cleavage patterns. The SF NPV genome was cleaved
into 8, 15, and 25 fragments by the restriction enzymes BamHI, Hindlll, and
EcoRl, respectively (Fig. 1). The estimated molecul ar weights of the
restriction fragments (see Table 6) have already been reported (manuscript
submitted). As described in a later section, slight corrections to the
molecular weights of BamHI fragments were made. It is seen that there are
22
-------
four sets of comigrating fragments in the EcoRI digest, one in the Hindlll
digest, and none in the BamHI digest. Thus, it was decided that the
construction of restriction maps for Hindlll and BamHI should be attempted
initially because they would allow less ambiguous assignment of transcription
patterns and regions of genetic variation between virus strains by using the
Southern blot hybridization technique.
E - x
o ? E
o .=
LU X m
Fig. 1. Cleavage patterns of SfNPV DNA
by the restriction endonucleases BamHI,
Hindlll, and EcoRI. The restriction
fragments were labeled with
[a-»*P]dATP in the presence of DNA
polymerase I. The partial fragment
between fragments D and E in the EcoRI
digest was the result of slightly
incomplete digestion.
F
G
H-
I'
J
K
Ml
l
Construction of a BamHI restriction map by cross-blot hybridization.
The first step in the mapping procedure was to determine whether there were
repeat sequences within the viral genome which could complicate the
interpretation of hybridization data. This was accomplished by the cross-blot
hybridization of cold BamHI fragments to 31P-labeled BamHI fragments (Fig.
2). Spots were found along the diagonal only. Thus, there were probably no
long repeat sequences present in the viral genome.
Partial digestion of SfNPV DNA should produce DNA fragments consisting of
two or more ''complete'' restriction fragments linked together. By
hybridizing JSP-labeled BamHI fragments to unlabeled, partially digested
BamHI fragments immobilized on a nitrocellulose sheet, the linkages between
different BamHI restriction fragments could be deduced (Fig. 3 and Table 1).
The deduction of the single linkages from fragments 3, 4, 5, and 6 are quite
obvious. Fragment 2 has four spots, indicating sequence homologies with BamHI
fragments B, E, F, and G. One of the two comigrating fragments must be
fragment B, because one spot is on the diagonal where the labeled and
unlabeled B fragments should intersect. Thus, the three BamHI fragments E, F,
and G must be linked together to form the other comigrating fragment. Since
23
Reproduced from
best available copy.
-------
fragment E is directly linked to both fragments F and G, the three fragments
must be linked in the manner indicated in Table 1, i.e., F-E-G. To sum up,
the data in Table 1 demonstrate linkages between five BamHI fragments, and the
deduced ordering is E-C-F-E-6.
24
-------
Hot BamHi
Fragments
A.
B-
G
H
**
c-
02
:£
a
I
o
X
oe
CO
<
ri
IT
f
JFigj._3.. Aut or adio graph of the patterns resulting from the cross-blot
hybridization between 3iP-labeled BamHI fragments and a cold partial BamHI
digest of SfNPV DNA. The J*P-labeled fragments of the partial digest was
run side by side with the cold fragments and transferred to the nitrocellulose
sheet to facilitate the identification of the unlabeled fragments.
Reproduced from
best available
copy.
25
-------
To complete the ordering of BamHI restriction fragments, J2P-labeled
Hindlll fragments were hybridized to nnlabeled BamHI fragments in another
cross-blot hybridization experiment (Fig. 4 and Table 2). If a Hindlll
fragment shares sequence homo logy with only one BamHI fragment as is the case
with fragments E, G, Ij, J, K, L, M, and N, the Hindlll fragment in question
mnst be entirely within the BamHI fragment involved. However, when a Hindlll
fragment shares sequence homology with two or more BamHI fragments, these
BamHI fragments must be linked together with the BamHI restriction sites at
the linkage points lying within the Hindlll fragment involved. This is
illustrated in Table 2 by the Hindlll fragments A, B, C, D, F, H, and Ij.
Thus, with BamHI fragment B linked to fragments H and D and BamHI fragment A
linked to fragments D and G, the ordering of the BamHI restriction fragments
could be completed (Fig. 5 a).
Construction of a Hindlll restriction map bv cross-blot hybridization.
Using the data presented in Table 2 in combination with the known ordering of
the BamHI restriction fragments, a partial ordering of the Hindlll fragments
could be deduced (Fig. 5b). Where uncertainties in ordering existed, the
fragments involved were included in one block. This is true for Hindlll
fragments E, G, and L, which lie totally within BamHI fragment A, Hindlll
fragments 1^ , and M, which lie within BamHI fragment D, and Hindlll
fragments K and N, which lie within BamHI fragment C.
To clear up these ambiguities, s*P-labeled EcoRl fragments were
hybridized to unlabelled Hindlll fragments in a cross-blot hybridization
experiment (Fig. 6 and Table 3). The linkage groups were deduced by the
reasoning described above. In the assignment of linkages deduced from
comigrating EcoRl fragments Cj_ and C2, it is seen that Hindlll fragments D
and E must be linked because fragments H and 1^ are known to be linked and
located well away from both D and E. However, the assignments involving EcoRl
comigrating fragments D^, D2, and 03 are more speculative. Fortunately,
the ordering of the Hindlll fragments involved is already precisely known.
With the additional information in Table 3, the Hindlll restriction map can
now be deduced. It is seen that Hindlll fragment E is linked to both
fragments D and L. Thus, it is easy to reason that th fragments D, E, F, G,
and L are joined in the order D-E-L-G— F. Similarly, knowing that fragment
*1 is linked to both fragments A and M, one can deduce that the fragments A,
F» Ii, and M are linked in the order F-M-Ii-A. Finally, with Hindlll
fragment K linked to fragment B, the fragments B, Ij, K and N can be
unambiguously joined as I2-N-K-B. The resulting restriction map for Hindlll
is shown in Fig. 5c.
Double digestion pj SfNPV DNA by BamHI and Hindlll. The double
digestion of SfNPV DNA by BamHI and Hindlll serves a threefold purpose.
First, it provides an independent confirmation of the restriction map deduced
from cross-blot hybridization. Second, it allows the precise alignment of
Hindlll restriction fragments within the BamHI map or vice versa. Last, it
gives better estimates of the molecular weights of large DNA fragments because
these fragments are cleaved by the second enzyme into smaller fragments that
are within the range of molecular weights provided by the markers. In our
case, this is applicable to the BamHI fragments A and B.
26
-------
TABLE 1. Cross-blot hybridization of ^P-labeled
BamHI fragments to unlabeled BamHI partial
digestion products
TABLE 2. Cross-blot hybridization of"P-labeled
Hindlll fragments to unlabeled BamHI fragments
Partial diges-
tion products
1
2"
3
4
5
6
7
8
9
10
11
12
13
BamHI fragments
with homologous
sequences
A
B
E, F, G
C, F
E, F
C, H
E, G
C
D
E
F
G
H»
H
Linkage groups
F-E-G
C-F
F-E
H-C
E-G
° Two comigrating fragments.
"Fragment 12 probably corresponds to BamHI
fragment H linked to a short fragment since fragment
13 is on the diagonal marking the intersection points
of identical cold and ^P-labeled BamHI fragments.
The short fragment was not observed in the 0.7%
agarose gels because its small size allowed it to migrate
ahead of the bromophenol blue marker.
Hindlll frag-
ment
A
B
C
D
E
F
G
H
1
j
K
L
M
N
BamHI fragments with
homologous sequences
B, D
C, E, F
E, G
A, G
A
A, D
A
B, H
A", C, D, H
B
C
A
A", D
C
Linkage groups
for BamHI
D-B
C-F-E
E-G
G-A
A-D
B-H
H-C
0 These two spots suggest a possible sequence ho-
mology between parts of BamHI fragment D and
BamHI fragment A. The relevant sequences within
BamHI fragment D are located near the junction
between Hindlll fragments M and Ii. The sequence
within BamHI fragment A is located within Hindlll
fragment L (Fig. 8 and Table 5).
G
I
*
3
Q.
09
0
3
Cold BamHI Fragments
AF ~D_
n A •*"•
DBc «.
1ST .
G ^
H -.
''J1'
KJ
M * •
N . *
•
FIG. 4. Autoradiograph of the patterns resulting
from the cross-blot hybridization between 32P-labeled
Hindlll fragments and cold BamHI fragments ofSF
NPV DNA. The smearing of the spots was due to the
slight overloading of the sample slots during get
electrophoresis of the cold BamHI fragments.
TABLE 3. Cross-blot hybridization of "P-labeled
EcoRI fragments to unlabeled Hindlll fragments
r, p. //mdlll fragments
fragment «*>> homologous se-
quences
A
B
C,
C2
D,
D2
D3
E
F
G
H
I
J,
K
L
M
N,
N,
0
P
Q
K
S
T
F, G,L
A, I
D, E, H, I
B, C, D, E
A
C
I, K, N
F
B
C,J
M
E, L
A
F, K
H, J
F
A,J
B, K
B
M, I
Linkage groups for
Hmdlll
L-G-F
Ii-A
D-E, H-I..
B-C, C-D, E
I2-N-K
E-L
J-H
A-J
K-B
M-I,
Reproduced from
best available copy.
27
-------
Hot ECoFU fragments
O
-toon D "a z
I/
c_ _
m
0
0CD>
**d
*•
fc»
i
•*>• j-
•j
X
>ro
p
m
Tl
D
I
- — .
C--
S1
O
o_
Q.
I
5"
a.
Tl
S
CO
3
(D
at
F i g . 6.. Aut or adio graph of the patterns resulting from the cross-blot
hybridization between 32P-labeled EcoRI fragments and cold Hindlll fragments
of SfNPV DNA.
SF NFV DNA was first digested with Hindlll and then with BamHI, end
labeled with [a-3*P]dATP, and electrophoresed in a 0.7% agarose gel. A
complete Hindlll digest was ran at the same time so that the fragments that
were cleaved by BamHI could be identified. The molecular weights and
stoichiometry of the restriction fragments were estimated as described above.
The BamHI and Hindlll double-digest patterns are shown in Fig. 7. It is seen
that the Hindlll fragments B, C, D, F, H, and "i^ have disappeared. In
addition, 13 new fragments are present. Referring to the restriction maps in
Fig. 5, this is exactly what is expected, with Hindlll fragments C, D, F, H,
and \i each being cleaved once and fragment B cleaved twice. Fragment A
should also be cleaved once. This is confirmed by its slight shift in
position to one corresponding to a lower molecular weight. The cleaved-off
piece of DNA is probably so short that it is running ahead of the bromophenol
blue marker and his migrated out of the gel. The estimated molecular weights
of these double-digestion products are presented in Table 4.
Alignment
alignment
of Hindlll
map. To achieve the
of Hindlll fragmgments within the BamHI map, J2P-labeled Hindlll
fragments were hybridized to unlabeled DNA fragments from a Hindlll-BamHI
double digestion in a cross-blot hybridization experiment (Fig. 8). The
J2P-labeled Hindlll fragment s will hybridize to the double-digest fragments
that share their sequence homology, thus allowing the identification of
specific double-digest fragments that form the original Hindlll restriction
fragments (Table 5). The location of BamHI sites within the Hindlll fragments
can then be calculated from the estimated molecular weights of the
28
Reproduced from
best available copy.
-------
double-digestion fragments listed in Table 4. The alignment of Hindlll
fragments within the BamHI map was thus accomplished. For example, the
Hindlll fragment D, made up of fragments 4 and 18 of the double digest, spans
the BamHI fragments A and 6. Since fragment 4 is too long to fit into BamHI
fragment G, it must be within BamHI fragment A, whereas fragment 18 lies
within BamHI fragment G. Similar deductions can be made, and the results are
summarized in the third column of Table 5.
During the initial alignment, it was found that the previously determined
molecular weights of BamHI fragments A and B were too low. Thus, using the
estimated molecular weights of the double-digestion fragments (Table 4), more
accurate values of 24.1 z 106 and 18.5 z 10<>, respectively, were assigned
to the BamHI fragments A and B and used in the construction of the final
restriction map of BamHI. The molecular weights of the other BamHI fragments
were proportionally adjusted so that the size of the whole DNA molecule was
again 82.5 z 10&. The molecular weights of the Hindlll and BamHI
restriction fragments used in the map are presented in Table 6. The
restriction map of SF NPV DNA for BamHI and Hindlll is shown in Fig. 9.
DISCUSSION
The restriction map of the SF NPV DNA genome is presented in Fig. 9 in
linear rather than circular form for the sake of simplicity. However, the
restriction enzyme mapping data confirm the circular nature of the DNA
molecule. The SF NPV genome consists of mainly unique sequences. This is in
agreement with the results from Kelly's reassociation kinetics ezperiments (7)
and our previous partial denaturation mapping data (J. Virol. 44: 747-751,
1982). Our cross-blot hybridization data (Fig. 4 and 8, Tables 2 and 5)
indicate that the only possible repeat sequences of any appreciable length may
be those located in Hindlll fragment L and those near the junction of Hindlll
fragments M and 1^ . However, one cannot ezclude the possibility of
additional short repeat sequences within parts of the genome where no BamHI,
Hindlll, or EcoRl restriction sites are present.
The restriction maps for BamHI and Hindlll sites should provide
sufficient sectioning of the SF NPV genome to facilitate initial studies of
transcription patterns and genetic variations between virus strains. The
relatively precise locations of the BamHI and Hindlll restriction sites are
confirmed by our double-digestion data where good placement of the sites for
the second enzyme within the sites of the first enzyme is possible. If a
specific region of the genome needs to be studied more closely, restriction
mapping with a third our fourth enzyme such as EcoRl or Xbal may be attempted
to provide an even smaller sectioning of the viral genome.
29
-------
TABLE 4. Molecular weights of Hindlll-BamHI
double-digest fragments
c
D
t.a.L
F
-:, M
A
j
H
't
«.N
I
c
0
t
L
S
F MM,
A
J
»
'•hr
•
FIG. 5. a, Physical map of SF NPV genome for
Bam HI; b, partial physical map of SF NPV genome
for Hindlll; c, physical map of SF NPV genome for
Hindlll.
=
ll 2
I X
m
—
C
13
17
Ac
Cr
G
H
J"'z
JK
L
M
N
21-
FIG. 7. Cleavage patterns of SF NPV DNA by
Hindlll and (Hindlll + BamHI). The restriction
fragments were labeled with fa-32P]dATP in the pres-
ence of DNA polymerase I.
Fragment
1
2
3
4
5
6
7
8
9
10 (2 comigrating fragments)
11
12
13
14
15
16
17
18
19
20
21
Mol wt (10°)
11.1
7.8
7.0
6.7
6.4
5.3
3.9
3.8
3.7
3.5
3.1
2.7
2.4
1.9
1.8
1.8
1.6
1.5
1.4
0.72
0.59
TABLE 5. Sequence homology between the Hindlll-
BamHI double-digestion products and Hindlll or
BamHI restriction fragments
Double-digest fragment
1
2
3
4
5
6
7
8
9
10
(2 comigrating fragments)
11
12
13
14
15
16
17
18
19
20
21
Hindlll
fragments
with ho-
mologous
se-
quences"
A
C
E
D
B
G
H
I
J
F
K
B
F
L
12
It
L'.M
C
D
N
B"
Hrf
BamHI
fragments
with ho-
mologous
se-
quences'
B
E
A
A
F
A
B
D
B
D
C
C
A
A
H
C
A', D
G
G
C
E
H
" From cross-blot hybridization data.
* From restriction map alignment.
' The spot at the point of intersection of fragment
16 and fragment L suggests a possible sequence ho-
mology between HmdIII fragments L and M (i.e.,
fragment 16 in the double digest). This is already
indicated in Table 2.
rf These are deduced by comparing the molecular
weights of complete HmdIII fragments and their dou-
ble-digestion products as well as restriction map posi-
tions.
30
-------
Hindlll, and EcoRI restriction fragments Reproduced from
k <» e » »U>tt*lA. •%!• /• « ^ v/ ^^%nwi^
Fragment
BamHI
A
B
C
D
E
F
HmdIII
A
B
C
D
K
F
G
H
I,
I.
1
K
I.
M
N
EcoRI
A
B
C,
D,
D,
K
F
<;
H
I
J,
•I,
K
L
M
N,
M.
0
p
Q
R
S
T
BamHI
» t i ii >S7fmfi
24.1
18.5
10.2
9.1
8.8
6.0
3.2
2.6
11.3
10.2
9.6
8.4
6.9
6.1 •"«— *
5.3 c * *
4.4
3.9 *» f •* '
3.9
3.6
35 am ^
2A f
1.8 £ = *
1.4 T 9 •
g *•» «?
1 »
8.1 +
7.1 : * »
6> I <"'*»'
6.1 5 ~ *
6.1
-------
REFERENCES
1. Bud, H.M. , and D. C. Kelly. 1977. The DNA contained by nuclear
polyhedrosis viruses isolated from four Spodoptera spp. (Lepidoptera,
Noctuidae): Genome size and configuration assessed by electron
microscopy. J. Gen. Virol. 37:135-143.
2. Burgess, S. 1977. Molecular weights of lepidopteran baculovirus DNAs:
Derivation by electron microscopy. J. Gen. Virol. 37: 501-510.
3. Greene, P.J. M. C. Bethlach. H.M. Goodman, and H.W. Boyer. 1974. The
EcoRl restriction endonucl ease s, p. 87-105. In: R.B. Wickner (ed.),
Methods in Molecular Biology, vol. 7. Marcel Dekker, New York.
4. Harrap, K.A., C. C. Payne, andJ.S. Robertson. 1977. The properties of
three bacnloviruses from closely related hosts. Virology 79: 14-31.
5. Jeffreys, A. J., and R.A. Flavell. 1977. A physical map of the DNA
regions flanking the rabbit p-globin gene. Cell 12: 429-439.
6. Jovin, J.M., P.T. Englund, and L.L. Bertsch. 1969. Enzymatic synthesis
of DNA. XXVI. Physical and chemical studies of a homogenous DNA
polymerase. J. Biol. Chen. 244: 2996-3008.
7. Kelly, D. C. 1977. The DNA contained by nuclear polyhedrosis viruses
isolated from four Spodoptera Sp. (Lepidoptera, Noctuidae): Genome size
and homology assessed by DNA reassociation kinetics. Virology 76:
468-471.
8. Knudson, D.L., and T.W. Tinsley. 1978. Replication of a nuclear
polyhedrosis virus in a continuous cell line of Spodopte ra
frugiperdaL: Partial characterization of the viral DNA, comparative
DNA-DNA hybridization, and patterns of DNA synthesis. Virology 87:
42-57.
9. Miller, L.K., and K.P. Daves. 1978. Restriction endonuclease analysis
for the identification of baculovirus pesticides. J. Virol. 35:
411-421.
10. Radloff, R., W. Bauer and J. Vinograd. 1967. A dye-buoyant density
method for the detection and isolation of closed circular duplex DNA:
The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A.
57: 1514-1521.
11. Richardson, C. C. , C. L. Schildkraut, H.V. Aposhiand and A. Kornberg.
1964. Enzymatic synthesis of deoxyribonuclei c acid. XTV. Further
purification and properties of DNA polymerase of E.coli. J. Biol.
Chem. 239: 222-232.
12. Rigby, P. W. , J.M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling
DNA to high specific activity in vitro by nick translation with DNA
polymerase I. J. Mol. Biol. 113: 237-251.
32
-------
13. Smith, G. E. , and M. D. Sumemrs. 1979. Restriction maps of five
Autographa cal if ornica MNPV variants, Trichoplusia ni MNPV, and
Galleria mellonella MNPV DNAs with endonucleases Smal, Kpnl, Bamfll.
SocI, Xhol, and EcoRl. J. Virol. 30: 828-838.
14. Smith, G.E., and M.D. Summers. 1980. Restriction map of Rachiplusia
hji .c.a.l.i.f.o.r.n.i.c.a and Rachiplus i a ou nuclear
polyhedrosis virus recombinants. J. Virol. 34: 693-703.
18. Wahl, G.M., M. Stern, and G.R. Stark. 1979. Efficient transfer of
large DNA fragments from agarose gels to diazobenzyloxymethyl- paper and
rapid hybridization by using deztran sulfate. Proc. Natl. Acad. Sci.
USA 76: 3683-3687.
19. Wilson, 6.A., and F. F. Young. 1975. Isolation of a sequence-specific
endonuclease from Bacillus amvloliquefaciens H. J. Mol. Biol. 97:
123-125.
33
-------
Section D: Construction of a Cloned Library
of the Hindlll DNA Fragments of Spodoptera
Frugiperda Nuclear Polyhedrosis Virus Genome and
Mapping of Novel Fragments
Yuan-Ming Wu1*, Eng-Chun Mar1, John J. Hamm2,
Clinton Kawanishi3, and Eng-Shang Huang1
J-Cancer Research Center and Departments of Medicine, Microbiology and
Immunology, University of North Carolina, Chapel Hill, North Carolina 27514.
'Southern Grain Insects Protection Agency, DSDA, Tifton, Georgia 31793 and
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.
ABSTRACT
Cleavage of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV)
Ohio Strain, DNA with restriction endonuclease Hindlll yields 18 fragments
ranging in size from 0.65 z 10<>, to 11.3 z 1C)6 daltons. Among these 18
fragments two with molecular weights of 0.65 z 10*, 0.93 z 10* and 0.68 z
10', respectively were not found previously. A cloned library of Hindlll
fragments of this strain was constructed using the pi asm id pBR322 and the
recipient bacterium Escherichia coli strain LE392. Blot hybridization was
used to determine the viral origin of the cloned inserts. The library is
representative of 100% viral genome. The nick-translated recombinant pi asm ids
were used to localize the map region of the three newly discovered Hindlll
fragments. The physical map of Hindlll SfNPV DNA fragment was confirmed.
INTRODUCTION
More than 400 baculovirus species were isolated from many host insects.
Only a few of these viruses have been used as biological pesticides in
agriculture and forestry. In the process of selecting and developing safe and
effective pesticidal viruses, information on the molecular biology and genomic
structure of baculoviruses have been obtained. Baculovirus genomes have been
analyzed with restriction endonucleases (11,12), and the restriction maps of
JLU_t.o.g.r.a.P.A§ californica NPV-DNA have been reported (4). It is ezpected
that genetic manipulation of baculoviruses will be reported in the near future
in attempts to develop improved viral pesticides.
SfNPV, a member of the baculovirus group, is one of the model systems for
the study of the molecular biology of insect viruses. It has a double
stranded, circular supercoiled DNA genome with a molecular weight of 80 z
10* (14,2,3,7). The characterization of the SfnPV genome and the physical
map for BamHI and Hindlll fragments of SfNPV genome have been published
previously (8,9). In this communication we report a cloned library of SfNPV
Hindlll fragments generated in the pBR322 vector system. These recombinant
plasmids are available for propagation of SfNPV DNA restriction fragments in
large quantity for molecular biological experiments. During the cloning of of
SfNPV Hindlll DNA fragments, three additional small DNA fragments were
uncovered. A revised restriction fragment map with the three additional DNA
fragments is presented.
34
-------
MATERIALS AND METHODS
JPu.r_iJ_i.c_a_t_i.on__2.f _vj.£JLJL_DNA_a.n.d_.p_l_a_sm_i.d_DN.A. The procedure for
purification of SfDNA has been previously described by Loh et al. (8,9).
The pi asm id pBR322 was extracted from bacteria by alkaline lysis as
described by T. Haniatis et al. (10) with some modifications. Briefly, the
bacterium E. coli LE3 92 containing pi asm id pBR322 was grown in 1 liter of M9
medium (Na2HP04 6g, KH2?04 3g, NaCl 0.5g, NH4C1 Ig and
supplement with final concentration of 0.1 mM CaCl2, 1 mM MgCl2, 0.2%
glucose, 0.12% casamino acid, 1 fig/ml of thiamine, 100 jig/ml of ampicillin) at
37°C on a shaker. Until the culture reached early log phase (O.D.600
= 0.6) 150 ug/ml of chl or amphenicol was added. The bacteria was incubated
overnight. The cell pellet was suspended in 10 ml of ice-cold solution
containing SO mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8) and 4 mg/ml
lysozyme. After 10 min, 20 ml 0.2N NaOH and 1% SDS was added and mixed
gently. For an additional 10 min on ice 16 ml of 3M Na-acetate solution was
added. The lysate was centrifuged on a Sorvall GSA rotor at 12,000 rpm
(23,000 g). The supernatant was extracted three times with phenol/chloroform
(1:1) and then ethanol precipitated. The supercoiled pi asm ids were further
purified by centrifugation to equilibrium in cesium chloride-ethidium bromide
gradient (density 1.56 gin/ml with 500 ug/ml of EtBr) .
Construction and transformation with recombinant plasmids. PI asmid
pBR322 contains a single Hindlll site, which interrupts the tetr acycl ine
resistance gene but does not affect the ampicillin resistance gene.
Hindlll-cl eav ed pBR322 was subjected to E. coli alkaline phosphatase
digestion at a concentration of 30 units per p mole of 5'-phosphate ends in 10
mM Tris-HCl, pH 8.0, at 65°C for 30 min to prevent sel f-liga tion of the
vector during the recombination reaction. Hindlll restricted viral DNA
fragments were ligated to the cloning vector by incubating 1 ug of SfNPV-DNA
fragments, 0.1 ug of pBR322 and 2 units of T4 ligase (BRL) for 18 hr at 12°C
in a ligation buffer (100 mM NaCl, 0.1 mM ATP, 0.25 mM MgCl2, 10 mM BTT, 50
ug/ml BSA, 50 mM Tris-HCl and pH 7.5) in a total volume of 15 ul.
Transformation of LE392 cells with recombinant plasmid was done according
to Dagert and Ehrlich (11). The strain LE392 was cultured in L-broth (1%
trypsin, 0.5% yeast extract, 0.5% NaCl pfl 7) at 37°C on shaker for 3-4 hr
until Ag5Q=0.2 was reached. After chilling on ice for 20 min, the culture
was pelleted at 3000 rpm (IEC Model HN-SII centrifuge) for 10 min. The
bacterial pellet was suspended in CAST solution (50 mM CaCl2, 10 mM NaCl,
and 25 mM Tris-HCl, pH 7.5) and held in ice bath without agitation. After 20
min the cells were recentrifuged and resuspended in a small volume of ice cold
CAST solution. Cells were used immediately for transformation.
To 0.1 ml of treated cells, 15 ul of ligation mixture containing
recombinant plasmid was added. After 10 min on ice the samples were incubated
at 42°C for 2 minutes, and at 37°C for 10 min in a water bath, and then
chilled in ice bath again. Two ml of L-broth were added and samples were
agitated at 37°C for 1 hr to allow plasmid gene expression. A sample of
each culture was spread on appropriate selection plates containing
antibiotics. The transformed colonies were screened by a hybridization
procedure as described by Grunstein and Hogness (6).
35
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Characterization of recombinant plasmids. Each recombinant colony
detected by hybridization against nick translated 32p SfNPV-DNA was
amplified in LB broth containing 100 ug/ml of ampicillin for further
restriction enzyme analysis. The rapid mini-prep screening procedure
described by Birnboirn and Doly was used to identify isolated recombinant
plasmids. Hindlll digested recombinant pi asm id and restriction fragments of
SfNPV-DNA were separated by el ectr ophor esis in 1% agarose gel in E buffer
(0.04M Tris-HCl pfl 7.2, 0.02M Na-acetate and 1 mM EDTA) . For critical
identification of viral DNA inserts, the restricted DNA fragments of
recombinant plasmids were end-labeled i,n v_it.r.o with Kornberg's DNA
polymerase. Each sample containing 5-30 ng DNA was incubated in 100 mM
Tris-HCl pH 7.5, 20 mM NaCl, 5 mM MgCl2, 6 mM 0-mer captoe thanol, 0.1 mM
dCTP, dGTP, TTP, 2 uCi [a32P] dATP and 0.5 unit polymerase at room
temperature for 5 min and then subjected to 1% agarose gel electrophoresis in
E buffer. After electrophoresis the gel was dehydrated by vacuum on a gel
dryer and exposed to Kodak X-Omat R x-ray film. DNA inserts of the
recombinant pi asm id were identified according to migrating distance of the DNA
fragments
The recombinant clones were also identified by Southern blot
hybridization. After electrophoresis the restriction fragments of SfNPV-DNA
were transferred onto nitrocellulose paper by the Southern technique (13).
Hybridization of nick-translated SfNPV DNA to Southern filter was accomplished
by incubating the filters at 65°C for 18 hr in 6xSSC, 50 ug/ml of yeast RNA,
50 ug/ml of calf thymus DNA, 25 ug/ml of polyA, 2.5 x Denhardt's solution and
0.5% SDS. After hybridization, filters were washed in 2xSSC for 1 hr at room
temperature, and then 3 times washing in O.lxSSC-0.1% SDS for 20 min at
55°C. The filter was air dried and then exposed to x-ray film.
RESULTS
Restriction enzyme Hindlll digest of viral DNA. As shown in Fig. 1 and
Table 1 the Hindlll digestion of SfNPV genome produces 18 fragments ranging in
size from 0.65 to 11.3 x 10', as estimated by comparing their mobilities in
1% agarose gel with standard phage XDNA fragments digested by Hindlll.
Hindlll DNA fragments 0, P and Q which were not identified in the previous
report (8) have been resolved in the present study by end labelling with
[
-------
hybridization shown. The linkages of Hindlll DNA fragments deduced are
' ' D-(E, L, G, )-F-(M, 0) Ix and A-J-H-Ia-( N, K)-B-( P, Q)-C-D' ' . Our
previous mapping results demonstrate the relative fragments order (E,L,G),
(M, 0) and (N,K), which indicates a DNA fragment order of "D-E-L^G-F-M-0-Ij
- A-J-H-Ij-W-K-B-(P,Q)-C-D'' .
tm i B r i i
Fi^. 1. Hindlll digestion fragment pattern of purified SfnPV DNA.
Restricted fragments were end-labeled with [a-'*P]dATP and separated by
electrophoresis on a 1% agarose gel. Th e additional DNA fragments 0, P and
Q were resolved by this method.
A BCD CF GH.», t, J * t/MNO'PQ
Fig. 2. Hybridization of cold BamHI-digested SfNPV DNA with a-»2P dATP
labeled recombinant plasmid with Hindlll F, I, M and 0 DNA inserts.
Reproduced from
best available copy.
37
-------
Reproduced from
best available copy.
tt
f !
JFLgJ_I. Hybridization of »*P labeled Hindlll DNA fragments F, Ix, M
and 0 to restricted SfNPV DNA (EcoRI. Bglll, and Xbal cleaved total DNA
subset). EcoRI, Bglll, or Xbal cleaved SfNPV DNAs were hybridized with [-
a-» *P]dATP-nick-translated SfNPV viral DNA or cloned F, I, M, and 0
fragments. As shown below, HindHI fragments F, 0 shared sequence homologies
with Bglll-digested SfNPV DNA.
The location of the M and 0 fragments were also simultaneously determined
by hybridization of IJP labelled M and 0 DNA fragments to whole sets of
SfNPV DNA fragments generated by EcoRI, Bglll and Xbal digestion (Fig. 3).
Due to the small size and close linkage of the 0 and P fragments, their exact
orientation has not been defined. Based on the intensity of hybridization to
BamHT E fragment, it is suggested that HindHI Q is located next to Hind C
fragment. The updated physical maps of the SfNPV BamHI and HindHI DNA
fragments is summarized in Fig. 4. The actual map should be circular.
Construction of the cloned library. The first cloning was attempted
with unfractionated, total restriction fragments of SfNPV DNA. The fragments
were ligated to the plasmid pBR322 and used to transform to E. coli LE392.
The bacterial colonies were screened on appropriate plates containing either
ampicillin or ampicillin plus tetracycline. Transfection with 0.1 (ig of SfNPV
DNA yielded 3000 colonies of ampicillin resistant and tetracycline sensitive
38
-------
(ArTs) phenotype recombinants. By colony hybridization 78 positive blots
were obtained from 986 colonies. Recombinant plasmids were analyzed by
el e ctrophor e ti c mobility. Thirteen recombinant plasmids with inserts
representing 60% of the genome. The remainder of 5 genomes were subcloned.
Hindlll cleaved viral DNA was subjected to electrophoresis on 1% agarose gel.
The desired fragments were cut from the gel and electrophoresed. After phenol
and chloroform extraction the DNA fragments were further cleaned by passing
through a RPC-5 analog mini-column (BRL) to get rid of snlfated
poly sa ccharides which are potent inhibitors of ligase. The purified
individual fragments were ligated to pBR322. In this way clones carrying the
remaining Hindlll DNA fragment inserts were obtained. Analysis of each of 18
recombinant plasmids by Hindlll enzyme is shown in Fig. 5 a and b.
Recombinant plasmids with inserts of more than one viral DNA fragment were
frequently obtained but are not shown.
Bam Hi i A i p i B |H| c i f \ e
HinD111 I 0 I E ,L,G, F flM,'l, A ,J,H,l2,N,K, B (,P|°) C
II
0 10 20
30 40 50 60 70 80
Molecular Weight ( 1 *106Daltons)
Fig. 4. Physical maps of the SfNPV genome for restriction endonucleases
Hindlll and BamHI. The actual map should be circular.
39
-------
Reproduced from
best available copy.
A Sf A B C D E F G H |j I2 J K LM- N- O PQ Sf
Fig. 5 a. The library of cloned recombinant plasmids. The recombinant
plasmid DNA was cut with Hindlll and subjected to electrophoresis on 1%
agarose gel. DNA was visualized by ethidium bromide stain. A,B,C....Q
represents individual recombinant plasmids. The band common to all the
digested plasmids is linear pBR322. =lambda DNA standard.
40
-------
;..•!.• H
nslated recombinant cloned DNA.
Reproduced from
best available copy.
41
-------
DISCOS SIGN
Due to the host specificity and the lack of in vitro infectivity of
occluded Sf nuclear polyhedrosis virus, the molecular biological study of this
virus has been somewhat hindered. To facilitate such research, we have cloned
the Hindlll restricted SfNPV DNA in pBR322. The size of DNA inserts in these
recombinants ranges from molecular weight of 0.65 to 11.3 million. The
recombinant clones pSFP and pSFQ, which carried the Hindlll P and Q inserts,
respectively, were somewhat difficult to obtain. Several ligations and
cloning experiments were done before stable recombinant clones were obtained.
There was no difficulty in generating recombinant clones with inserts of more
than 15 Kb such as the largest Hindlll fragment of SfNPV ONA. During the
selection and identification of the recombinants clones, inserts possessing
small deletions were found. Such recombinant clones were problematic because,
like complete inserts they were ampicillin resistant, tetracycline sensitive
and hybridized to the original SfNPV DNA fragments. The mechanism of deletion
in these recombinants and the most frequent deletion site(s) remains unknown.
and pSFI2 carry nearly identical sized fragments of
about 3.9 Kb. Hybridization of »*P-labelled DNA of pSFIi and
pHD20I4 to SfNPV BamHI DNA indicates that they are two distinct DNA
fragments (Fig. 2). Separation of these two fragments could be achieved only
through cloning.
Hindlll DNA fragments 0, P and Q were overlooked previously because of
over electr ophor esis and loss of the small DNA fragments. By end labelling of
the SfNPV DNA fragments and shortening the el ectr ophor esis, the three
additional DNA fragments were discovered. The location of the P and Q
fragments were mapped to between the Hindlll B and C fragments. Presently we
do not have conclusive data to determine if the actual order of P and Q is
-B-P-Q-C or -B-Q-P-C-. Based on the hybridization of »*P Hindlll DNA
fragments B, C, P and Q to BamHI E fragment (as shown on Fig. 2) is believed to
be located next to Hind C fragment and that the former order ( -B-P-Q-C) is the
correct one. The DNA fragment map shown in Fig. 4 is linear but the data
indicates that the actual map is circular.
42
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TABLE 1
Molecular Weights of SfNPV DNA Fragments Cleaved by Hindlll ajid
Recombinant Clones Carried Corresponding DNA Fragments
HINDIII SfNPV
DNA FRAGMENTS
A
B
C
D
E
F
G
H
II
12
J
K
L
M
N
0
P
Q
MOL. WT. (10«) OF
DNA FRAGMENTS
11.3
10.2
9.6
8.4
6.9
6.1
5.3
4.4
3 .9
3.9
5.6
3.5
2.4
1.8
1.4
0.93
0.68
0.65
RECOMBINANT CLONES WITH
SfNPV DNA INSERTS
pSFA
pSFB
pSFC
pSFD
pSFE
pSFF
pSFG
pSFH
pSFIl
pSFl2
pSFJ
pSFK
pSFL
pSFM
pSFN
pSFO
pSFP
t>SFQ
TOTAL
86.96
43
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TABLE 2
Southern Blot Hybridization of 32P labelled
Hindlll Fragments to Unlabelled BamHI Fragments
and the Linkage Hap of DNA Fragments
Hindlll DNA
Fr a gment
(P»*P Labelled)
A
B
C
D
E
F
G
H
II
12
J
K
L
H
N
0
P
Q
BamHI Fragments with
Homologous Sequences
B
C, F, E (less intense)
E,G
A, G
A
A,D
A
B, H
D
C,H
B
C
A
D
C
D
E
E
Linkage Groups Deduced
for Hindlll Fragments
A-J-H
Ia-(N,K)-B
B-(P,Q)-C
C-D
D-(E,L,G)-F
D-(E,L,G)-F-(M, 0, I)
D-(E,L,G)-F
A-J-H
F-(M, O)-!1
H-I2-(W, K)-B
A-J-H
I*-(N.K)-B
D-(E, G, L)-F
F-(M, 0)-Ii
Ia-(N,K)-B
F-(0,M)-I*
B-(P,Q)-C
B-(P,Q)-C
44
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REFEREN CES
1. Birnboim, E.G. and J. Doly. (1979). A rapid alkaline extraction
procedure for screening recombinant pi asm id DNA. Nucleic Acids Res. 7:
1513-1520.
2. Bud, H.H. and D. C. Kelly. (1977). The DNA contained by nuclear
polyhedrosis viruses isolated from four Spodoptera spp. Lepidoptera
Noc tuidae) ; Genome size and configuration assessed by electron
microscopy. J. Gen. Virol. 37: 134-143.
3. Burgess, S. (1977). Molecular weights of Lepidopteran baculovirus
DNAs: Derivation by electron microscopy. J. Gen. Virol. 37: 501-510.
4. Cochran, M. A., E.B. Carstens, B.T. Eaton and P. Faulkner. (1982).
Molecular cloning and physical mapping of restrictiion endonuclease
fragments of Autographa californica nuclear polyhedrosis virus DNA.
J. Virol. 41: 940-946.
5. Dagert, M. and S.D. Ehrlich. (1979). Prolonged incubation in calcium
chloride improves the competence of Escherichia coli cells. Gene 6:
23-28.
6. Grunstein, M. and D. Hogness. (1975). Colony hybridization: A method
for the isolation of cloned DNAs that contain a specific gene. Proc.
Natl. Acad. Sci. USA 72: 3961-3965.
7. Knudson, D. L. and T.W. Tinsley. (1978). Replication of a nuclear
polyhedrosis virus in a continuous cell line of Spodoptera
frugiperda; Partial characterization of the viral DNA, comparative
DNA-DNA hybridization, and patterns of DNA synthesis. Virology 87:
42-57.
8. Loh, L.C., J. Hamm and E.-S. Huang. (1981). Spodoptera frugiperda
nuclear polyhedrosis virus genome: Physical maps for restriction
endonuclease BamHI and Hindlll. I. Virol. 38: 922-931.
9. Loh, L. C. , J.J. Hamm, C. Kawanishi, and E.-S. Huang. (1982). Analysis
of the Spodoptera frugiperda nuclear polyhedrosis virus genome by
restriction endonuclease and electron microscopy. J. Virol. 44:
747-751.
10. Maniatis, T. , E.F. Fritsch, and J. Sambrook. (1982). Molecular
Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, pp.
90-91.
11. Smith, G.E., and Summers, M.D. (1978). Analysis of baculovirus genomes
with restriction endonucleases. Virology 89: 517-527.
12. Smith, G.E. and Summers, M.D. (1979). Restriction maps of five
Autographa californica MNPV variants. J. Virol. 30: 828-838.
45
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13. Southern, E. M. (1975). Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Hoi. Biol. 98: 503-517.
14. Summers, H.D. and D.L. Anderson. 1973. Characterization of nuclear
polyhedrosis viral DNAs. J. Virol. 12: 1336-1346.
46
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Section E; Interaction of Spodoptera frugiperda NPV
With Various Mammalian Cell Lines in vitro
ABSTRACT
Detectable viral gene expression and viral nucleic acid synthesis were
not observed in SfNFV or viral DNA treated mammalian cell lines, including
diploid WI-38 human fibroblasts, EEL cell, NIH3T3 cells and guinea pig
embryonic fibroblast. Positive expression of SfNPV antigens were demonstrated
in virus infected homologous host SF 140AE cells. No positive transformation
of human fibroblasts by SfNPV and its DNA were found.
INTRODUCTION
The interaction of S. frugiperda NPV (SfNPV) with various mammalian
cell lines was studied by (a) viral structural antigens expression in virus
infected or viral DNA trnsfected permissive insect cell line (S. F. cell line
140 AE and IPLB, 21AE) and non-permissive various mammalian cell lines, (b)
viral DNA synthesis by SH cRNA-DNA membrane hybridization, (c) viral mRNA
synthesis by in situ 3*P DNA-RNA hybridization, and (d) the oncogenicity
of viral DNA by transformation of human fibroblasts (WI-38 and EEL), mouse
cell 3T3 and hamster embryonic cells with SfNPV DNA by calcium phosphate and
DMSO treatment. To date, we have not been able to demonstrate SfNPV viral
gene expression in non-permissive mammalian cell lines using either virus from
hemolymph of infected larvae or purified DNA. In contrast, positive results
were found with permissive S.F. 140 AE cells. Rapid degradation of
transfected DNA was found in SfNPV DNA transfected WI-38 cells.
MATERIALS AND METHODS
Cells. The human diploid WI-38 fibroblast and the EEL embryonic lung
cell lines were obtained from ATCC and Dr. F. Rapp, respectively.
Two additional mammalian cells, NIB 3T3 mouse cell line and guinea pig
embryonic fibroblast were also used for the infection and gene expression
studies. The cells were grown in D-MEM supplemented with fetal calf serum (6
to 10%), penicillin and streptomycin at 100 units and 100 |ig per ml,
respectively.
Insect cell lines. Spodoptera frugiperda (Strains 140AE and IPLB,
21AE), were grown in Bank's medium with 10% fetal calf serum at 28°C.
Virus strains. Three {J. frugiperda NPV strains (Ohio, Georgia and
North Carolina) were used for this study. The Ohio strain was used
predominantly for the DNA characterization. The virus used for infection came
from the hemolymph of virus infected Sf larvae.
Synthesis of viral complementary RNA (cRNA) The viral complementary
RNAs (cRNAs) were synthesized and labelled with *B UTP as described
previously (3).
47
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Radioisotope labeling of viral DNA in vitro with Kornberg's enzyme.
For in sitn »H or »*P DNA hybridization to detect viral specific mENA,
the nick translation method was used. This method for labelling with *H TIP
or **P XTP was described previously for human cy tome gal ovirns system. The
specific activity of DNA obtained was abont 2 x 10 * cpm/ng for *H and
1-2 x 108 cpm/iig for **P. Before being used as a radioactive probe,
each in vitro enzymatic labeled product was characterized for specific
activity and sensitivity (percentage) of DNA to Si nuclease (native or
denatured DNA). DNA preparations with low specific activity, uneven labeling
and high background (if more than 5% of DNA is resistant to S^ digestion
when denatured or sensitive to S^ digestion when it is in native state) were
not used as probes for reassociation kinetics analysis.
Complementary RNA-DNA hybridization.
(i) Membrane filter. The hybridization of in vitro synthesized
virus specific 3H-cRNA to immobilized denatured DNA was achieved by the
method of Gillespie and Spiegelman (2) as described by Huang et al. (3).
(ii) Cy tohybr idization in situ. This was carried out according to
the procedure of Gall and Pardue (1) with modifications. Cells were
hypotonically treated with either 0.1 x Hanks's solution or 0.1 x SSC for 20
min at 37°C and fixed with ice-chilled ethanol and acetic acid (3:1) for 10
minutes. The slide was then dipped in absolute alcohol three times,
alkalinized in 0.07 N NaOH for 3 min, and washed by dipping in 70 and 95%
alcohol three times each. 0.1 ml of viral specific *H-cRNA solution
containing 5 x 10s cpm was applied and covered with a cover glass and the
samples kept wet with 6 x SSC. The hybridization was carried out at 66°C
for 20 to 22 hours. After RNase digestion, complete washing and dehydration
with alcohol, the slides were dipped in NIB emulsion (Eastman) and air-dried
in a darkroom for at least 2 hours. For detection of viral specific mRNA, in
vitro nick translated **P viral DNA was used (S.A. 1 x 10s cpm/jig).
The cells were not alkalinized; but instead were treated with proteinase K
to uncover viral RNA. The hybridization was performed under the same
hybridization condition as before. Excess J1P DNA was removed by extensive
washing with 6 x SSC and heating at 70°C for 10 min to remove unhybridized
radioactive DNA. The aut or adiography was carried at -20°C for periods of 1
to 2 days up to 4 weeks, depending on the specific radioactivity of the
*H-labeled material after preparation as follows: The emulsion-cover slide
was dipped into dioxane fluid with PPO (1% w/v) and POPOP (0.2%) for
exposure. The cover slip or slide were then developed with Kodak D-19, fixed
with rapid fix, and stained with giemsa for 30 min. For detection of viral
messenger RNA in virus transformed cells, 3aP viral DNA or JH cRNA was
use d.
(iii) Transfection with viral DNA. The successful transfection and
transformation of human embryonic fibroblast with human CHV DNA fragments in
our laboratory encouraged us to study the possibility of oncogenicity of
pesticidal virus DNA and the additional possibility of pesticidal virus gene
expression in mammalian cells. The optimal conditions (.eg., concentration of
viral and carrier DNA used, concentration of DMSO, etc.) were evaluated and
established during the developmental stages of study in the permissive or
homologous cell systems, and then extended to mammalian and human cells.
48
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The following is the outline for the DNA transfection procedure which is
essentially that of Stow and Wilkie (6). Intact or NPV DNA fragments were
mixed with carrier salmon sperm DNA to a final concentration of 20 (ig/ml in
Hepes buffered saline containing 0.7 mM Na2HP04. The CaCl2 solution was
added to the DNA solution to a final concentration of 1.25 mM. The mixture
was overlayed on the pre-confluent cell culture (80% confluence). Thirty
minutes after overlaying the DNA solution, an additional 10 ml of MEM (for
WI-38, HEL and 3T3) or Grace's (for SF cell lines) media containing 4% fetal
calf serum was added. Four hours later, the transfected cell cultures were
subjected to DMSO shock (15% for 5 minutes). The cultures were then
maintained in MEM or Grace's media with 2 to 4% fetal calf serum for a period
of time. Viral gene expression was detected by the methods listed in the
previous sections. Kinetics of viral DNA synthesis was examined with specific
viral 3H cRNA-DNA hybridization on nitrocellulose membrane as described
previously (Huang and Pagano, 1977). Localization of infected or susceptible
cells was approached by *H cRNA-DNA in situ cytohybridization and
immunofluorescence test.
RESULTS
Resistance of mammalian cells to SfNPV infection. Anti-SfNPV serum
obtained from an immunized guinea pig was used for the detection of virus
specific antigen expression in SfNPV infected cell cultures by the indirect
immunoflucrescsnce test (IFA). Negative IFA was found in all four mammalian
cell lines infected with virus from the hemolymph of infected insects. In
contrast, viral antigen expression was readily detectable in the insect cell
lines, IPLB 21AE, and SF 140AE cells, infected with virus from hemolymph, (see
Figure 5-1-a). Under the phase contrast microscope, viral polyhedra were seen
within insect cells where virus specific immunofluorescence was also found.
(Figure 5-1-b).
Strain SF140AE insect cell lines and WI-38 human fibroblasts were used
for the DNA transfection study. Monolayer cultures were infected with
supercoil SfNPV DNA by the calcium phosphate precipitation DMSO method. At
4-7 days after transfection, the cell cultures were examined for viral antigen
by IFA and viral gene transcription by in situ DNA-mRNA cy tohybr idiz ati on.
Positive infected cells (about 1 in 104) were found in DNA transfected S.F.
140AE cells as detected by in situ cytohybridization using 3*P ATP
labeled SfDNA as probe (for detecting RNA). However, we were not able to
detect the positive transfection in WI-38 human fibroblast; either by FA or
in _si.L3 cytohybridization. Because the transfection efficiency in the
permissive cell was very low, it is certainly inappropriate to conclude that
there is no viral gene expression in human fibroblasts.
The standard focus forming transformation assay was also carried out with
SfNPV-DNA transfected WI-38, mouse Balb 3T3 and hamster embryonic cells. No
morphologically transformed cell lines were established. The application of
phorbol ester (TPA) at 20 ng/ml to DNA transfected culture did not enhance
or cause morphological transformation of human fibroblasts.
49
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Three- to four- day-old mouse-embryos were transfected with SfNPV-DNA
using calcium chloride method, and reimpl anted in fostered mothers for further
embryonic development. From three experiments with more than 48 transfected
embryos, only one normal baby was obtained. Unfortunately, in the control
group, where identical procedures were used (calcium phosphate-DMSO) less than
10% (2 out of 30) embryos developed into full term babies. Therefore the
failures in successful implantation and full-term gestation may be due to the
calcium chloride and DMSO treatment.
Fate of transfecting SfNPV DNA. The fate of transfected SfNPV DNA in
WI-38. guinea pig embryonic fibroblast and Sf 140 AE cells were further
examined and quantitated by membrane cRNA-DNA hybridization. Cells in 75
cm2 flask were transfected with 2 ug per flask of SfNPV DNA by the calcium
chloride-DMSO procedure (6). Cells were lysed with 1% sodium lauryl sulfate
(SDS) in 0.05M Tris-HCl, pB 9 and 0.01M EDTA at various times after
transfection. DNA was extracted and immobilized on nitro-nitrocellulose for
hybridization. Figures 4-2 show the relative quantity of viral DNA from
various transfected cells as reflected by counts of *H cRNA hybridized. In
SfNPV DNA transfected WI-38 and GP embryonic cells the input DNA was degraded
early and remained at the background level 3-4 days after infection. In
contrast, viral DNA synthesis was detectable at 72 hours after tr ansf ection in
insect SF140AE cells and gradually reached a plateau at a relatively low level
at 7 days.
DNA tr ansf ection offers the advantage of bypassing the membrane receptor
barrier. However, the tr ansf ection efficiency of DNA is rather low compared
to normal infection by intact virions. The low level of DNA synthesis in
SfNPV DNA transfected SF140 AE cells might be caused by DNA degradation as
well as inability to generate reinfection by progeny virus. The fate of
transfected SfNPV DNA was also studied by alkaline sucrose gradient
sedimentation. Extensive degradation into small DNA fragment was found
particularly in SfNPV DNA transfected WI-38 and guinea pig embryonic
f ibroblasts.
CONCLUSION
Numerous in v.i.t.r.g and in vivo experiments have been done to show
the species specificity and safety of the pesticide viruses. Although reports
suggest the low infectivity to vertebrate cells (Mclntosh and Kimura, 1974),
we were not able to demonstrate replication and expression in SfNPV
virion-infected and DNA transfected mammalian cells by DNA hybridization and
immunofluorescence tests. Intact SfNPV DNA was used for morphological
transformation of human fibroblast, in the presence and absence of tumor
promoters, but no evidence of transformation was obtained. In view of the
structure of nuclear polyhedrosis virus where virions are imbedded in a
crystalline protein matrix, the occurrence of permissive infections of
mammalian cells should be extremely rare. The nonpermissiveness of SfNPV
infection of mammalian cells as demonstrated in this study further support the
safety of SfNPV for use in controlling its pest host in agriculture.
50
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fig. Infection of Sf 140 AE cells with SfNPV from the **••**$
itfttt.d larvae. (a) phase contrast microscopic observat
immunof Increscent focus.
Reproduced from
best available copy.
-------
2 -
n
I
O
X
Q_
O
X
CO
1 -
24
48 72 96 " 144
hours post infection
168
Fig. 3. Transfection of Sf 140AE cell (•), WI-38 (A), and
embryonic fibroblast ( O) with SfNFV DNA. The DNA synthesis was
membrane hybridization nsing *H labeled cRNA of SfNFV MA.
52
guine a pig
detected by
-------
REFERENCES
1. Gall, J.-G. and M.L. Par due. (1971). Nucleic acid hybridization in
cytological preparation. Methods Enzymol. 24: 470-480.
2. Gillespie, D. and S. Spiegelman. (1965). A quantitative assay for
DNA-RNA hybridization with DNA immobilized on a membrane. J. Mol. biol.
12: 829-842.
3. Huang, E.-S., S.T. Chen and J.S. Pagano. (1973). Human
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