Ecological Research Series
CHARACTERIZATION OF SHRIMP BACULOVIRUS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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CHARACTERIZATION OF SHRIMP BACULOVIRUS
by
Max D. Summers
The Cell Research Institute
and
Department of Botany
The University of Texas at Austin
Austin, Texas 78712
EPA Grant R-803395
Project Officer
John A. Couch
Gulf Breeze Environmental Research Laboratory
Gulf Breeze, Florida 32561
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
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DISCLAIMER
This report has been reviewed by the Gulf Breeze Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
i i
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FOREWORD
The protection of our aquatic resources from damage caused by chemical
or biological pesticidal agents requires that regulations controlling the
use of specific agents in pest control be formulated on a sound scientific
basis. Accurate information on the novel biological control agents and
related biological entities being developed for use as pesticides is needed
to insure their efficacy and safety. The Environmental Research Laboratory,
Gulf Breeze, contributes to this need through research aimed at determining
precise identification, isolation, and characterization of biological control
agents and related species found naturally in aquatic organisms. The recent
finding by Gulf Breeze researchers of the first Baculovirus naturally
occurring in commercial shrimp illustrates the need to better understand the
nature of this large assemblage of arthropod viruses, some of which are under
development as biological control agents.
Thomas W. Duke
Laboratory Director
ill
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ABSTRACT
The research undertaken involved the partial characterization of a bacu-
lovirus of the pink shrimp, Penaeus duorarum. The significance of the study
is related to the fact that the shrimp baculovirus is morphologically similar
to insect baculoviruses which were considered unique to insect arthropods
prior to the discovery of shrimp nuclear polyhedrosis baculovirus (NPV).
Further, insect baculoviruses are being developed and applied as microbial
pesticides for the control of certain agricultural insect pests. Whereas
the baculovirus diseases in pests of agricultural or medical importance are
considered a desirable relationship, a baculovirus infection in shrimp is an
undesi rable one.
Research included investigations of the biochemical, structural, and,
where appropriate, biological properties of the shrimp virus as compared to
those of known and characterized properties of insect baculoviruses, both
granulosis and NPVs.
Evidence for any structural relatedness of the shrimp NPV to insect NPVs
has been confirmed in cross-reactions of purified shrimp NPV polyhedrin and
infected shrimp tissues to insect baculovirus antisera.
This report was submitted in fulfillment of Contract No. R-803395 by
the University of Texas, Austin, Texas, under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period September
23, 197^ to December 31, 1976.
IV
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CONTENTS
Foreword i i i
Abstract iv
Figures vi
Tables vi
Abbreviations and Symbols vii
1. Introduction 1
2. Conclusions 3
3. Recommendations 6
4. Materials and Methods 7
Selection and collection of infected organisms .... 7
Purification of occluded Pd^NPV 7
Solubi1ization of occluded virus 9
Amino acid analysis 9
SDS-polyacrylamide gel electrophoresis 9
Purification of enveloped nucleocapsids 10
Cell culture studies 10
DNA extraction 10
DNA spreading and electron microscopy 10
Immunodiffus ion 11
Competition radioimmunoassay (RIA) 11
5. Results and Discussion 12
Purification of polyhedra 12
Purification of enveloped nucleocapsids from
infected tissue 14
SDS-polyacrylamide gel electrophoresis 14
Amino acid analysis 17
Cell culture studies 17
PdlSNPV DNA 19
Serology 30
Immunodiffusion assays 30
Radioimmunoassay (RIA) 32
References 34
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FIGURES
Number Page
1 Isopycnic banding of shrimp virus polyhedra in sucrose
gradient 13
2 Purification schematic for fractionation of infected shrimp
hepatopancreatic tissue 15
3 SDS-polyacrylamide gel electrophoresis of shrimp polyhedrin
relative to insect baculovirus polyhedrins 16
4 Equilibrium banding of shrimp baculovirus DNA in ethidium
bromide-cesium chloride gradients 20
JfA T. bacteriophage DNA 20
AB Shrimp baculovirus DNA 20
5-10 Kleinschmidt preparations of shrimp baculovirus DNA 21
11 Immunodiffusion of shrimp polyhedrin 31
12 Immunodiffusion of infected shrimp hepatopancreatic tissue ... 31
13 Analysis of shrimp polyhedrin 33
TABLES
Number Page
1 Screening and Collection of Infected Shrimp 8
2 Amino Acid Analysis of Granulins and Polyhedrins 18
3 Deoxyribonucleic Acids of Baculoviruses 27
vi
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
AcMNPV
BSA
ccDNA
CPE
DAS
dlDNA
DNA
ETDA
GV
ug
mg
ml
mM_
NP-40
NPV
PdSNPV
rcDNA
RIA
rpm
SDS
SDS-PAGE
SSC
TN-368
TN-368-10
UV
SYMBOLS
c
hr
M_
S
-- Ajjtographa califormca nuclear polyhedrosis virus
-- bovine serum albumin
-- covalently closed DNA
cytopathic effect
dilute alkaline saline, pH 10.9
-- double-stranded linear DNA
deoxyribonucleic acid
ethylenediamine tetraacetic acid
granulosis virus
-- microgram
-- milligram
-- mill filter
-- millimolar
nonident 40
nuclear polyhedrosis virus
-- ^enaeus duorarum nuclear polyhedrosis virus
relaxed circular DNA
-- radioimmunoassay
revolutions per minute
sodium dodecyl sulfate
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
-- standard saline citrate
Trjchoplusia n? 368 cells
Clone 10, Trichoplusia nj^cells
ultraviolet
Angstroms
Celsius
-- hour
many nucleocapsids per envelope
-- enveloped single nucleocapsid
VI I
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SECTION 1
INTRODUCTION
Enveloped rod-shaped DNA viruses which are occluded in proteinic crystals
have been discovered and described as viruses unique for insect arthropods.
Routinely these viruses are described as nuclear polyhedrosis (NPV) and
granulosis viruses (G V) (Wildy, 1971) and more recently reclassified in
Baculoviridae. Baculovirus cytopathology and ultrastructure have been studied
extensively and until recently it was considered that they were unique in
that they were the only viruses known to be occluded and to be specific for
insect hosts. Now it is known that this is not the case (Vblkman et al.,
1976). Recent reports have shown that occlusion does not necessarily occur
for a baculovirus during all stages of the biological cycle. That is, it
may exist in both the occluded and nonoccluded states during the infection
sequence. The discovery of a baculovirus of the pink shrimp, Penaeus
duorarum (Couch, 197**; Couch et al,, 1975), has now extended the host range
for this class of viruses into the class Crustacea. Another report of a
possible baculovirus cites infection of the nuclei of hemocytes and connec-
tive tissue elements of the European crab Caj^cjmus maenas (Bonami , 1976).
"lore recently, a baculovirus of the blue crab Callinectes sapidus was dis-
covered in the epithelium of hepatopancreatic eel Is (Johnson, 1976). The
virus was observed in both juvenile and adult crabs of both sexes that had
been collected in the Chincoteague Bay, Virginia, and in Chesapeake Bay,
Maryland, and its tributaries. In one group of crabs sampled in the Chinco-
teague Bay, $2% were reported infected.
The significance of the observations of baculoviruses in noninsect
arthropods leads to a great deal of curiosity about any relatedness to
insect baculoviruses. This is especially significant from the standpoint of
the use of insect baculoviruses as biological pesticides, a development
which has recently resulted in the registration of two insect baculoviruses
for control of insect pests. Furthermore, characterization of the shrimp
baculovirus will aid in providing technology for monitoring in order to
study any effect that the virus may have upon populations of shrimp in
nature and the extent of prevalence. The latter is particularly important
from the standpoint of understanding the direct effect of the virus on its
natural host (Couch et al., 1975). There is direct evidence that severe
cytopathological effects occur in hepatopancreatic cells of infected
organisms. There is additional evidence that the virus may cause epizootic
mortalities in feral shrimp and larval shrimp in culture. Further, it is
important to understand what interactions may occur with stress involving
chemical pollutants. Couch et al. (1975, 1976, 1977) reports that poly-
chlorinated biphenyls may increase the prevalence of patent virus infections
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in experimental animals. The role that pollutants may play as stressors can
be effectively studied only through the use of quantitative and less ambi-
guous means of evaluating virus infections.
Lastly, it is important to study host specificity with regard to baculo-
viruses in more definitive detail. This is especially important relative
to the proposed role and use of insect baculoviruses as pesticides. Although
there appears to be little danger involved in the use of present baculo-
viruses as viral pesticides, the problem of host specificity will require
a more comprehensive definition. The discovery of baculoviruses in marine
crustaceans from different areas of the world suggests that a restricted
virus in terms of specificity for only insect arthropods is not now as
absolute as previously thought.
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SECTION 2
CONCLUSIONS
Studying the shrimp baculovirus in its natural environment and attempting
to recover sufficient quantities for characterization studies were difficult
during these studies and definitely limited the magnitude of effort and the
extent of the results obtained.
The shrimp baculovirus has chemical and physical properties which, al-
though similar to insect baculoviruses, are distinctly different. One of the
major problems was our inability to use routine dilute alkaline saline
solubi1ization in order to disaggregate the shrimp polyhedrin from its
crystalline form, and at the same time release enveloped nucleocapsids.
Ionic and nonionic detergents were required for the crystal solubi1ization
process and thereby the physical integrity of the enveloped nucleocapsid was
destroyed.
Attempts to recover enveloped nucleocapsids directly from infected
shrimp tissue were only partially successful in that only very limited
amounts of virus could be recovered after isopycnic banding on sucrose
gradients. Therefore, attempts to continue work with that form of the virus
for comparison with insect baculoviruses were not continued.
When comparing enveloped nucleocapsid structure and occlusion in a pro-
tein crystal, the general ultrastructural relationships are similar to those
routinely observed for insect baculoviruses. However, there are some
dissimilarities. The lattice spacing, and therefore the unit structure of
the crystal, is significantly larger and the polyhedral membrane is absent.
Since enveloped nucleocapsids could not be recovered in sufficient
quantities, no structural studies were done on that form of the virus.
However, SOS-PA-! E of shrimp polyhedrin demonstrated that it was significantly
larger as compared with insect baculovirus granulins and polyhedrins. On the
surface this might be considered evidence of unrelatedness since the insect
baculovirus polyhedrins and granulins have shown remarkable similarity in
size and composition. Summers and Smith (1976) demonstrated by two-dimen-
sional high voltage electrophoresis and peptide mapping that, although highly
purified insect baculovirus polyhedrins and granulins demonstrated some simi-
lar peptides and therefore similar primary structures, there were different
peptides indicating that each protein (in association with its enveloped
nucleocapsid component) was specific for that virus.
Serological studies published to date have revealed cross-reactions of
antisera and antigens for this class of baculovirus proteins. Initial
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attempts to compare shrimp polyhedrin by immunodiffusion were negative;
therefore, we considered that the protein was not related to the insect
baculovirus structural proteins. However, denaturing agents, such as SDS
used in the appropriate concentration so as not to introduce artifact, have
opened the conformation of the shrimp polyhedrin and revealed some cross-
reaction with insect baculovirus polyhedrin antisera. In particular, a
cross-reaction with AcMNPV polyhedrin and TnGV granulin and antisera was
significant and confirms that similar primary sequence or antigenic sites
do exist in shrimp and insect virus polyhedrins.
These observations may lead to considerable speculation concerning the
evolution of this class of unique proteins in association with virus infec-
tions. The nature and the possibility of this relatedness, however, should
not be considered a serious topic for speculation until more complete studies
have been conducted.
The shrimp baculovirus DNA, in terms of its structural properties and
size, very definitely is similar to insect baculovirus DMAs. The relative
ratio of ccDNA relative to rcDNA and dlDNA are remarkably similar. However,
additional information on relatedness cannot be provided at this time
because physical properties or information such as base-ratio analyses are
not adequate. At first it was not thought possible to utilize any one tech-
nique for comparing the shrimp DNA with other baculovirus DNAs because only
very small quantities could be obtained. With improved use and application
of 125I-label ing of nucleic acids, it is now possible to conduct physical
mapping experiments with restriction endonuclease enzymes. Gene segments
with similar base sequences could or should be revealed by this technique;
such studies are contemplated for future investigations.
The preliminary studies in invertebrate cell culture were entirely in-
adequate and were merely probes or superficial attempts to see if the shrimp
virus could be induced to replicate in vjj.ro. However, all of those studies
are now difficult to evaluate because it is known that polyhedra formation
is not considered a reliable indicator of virus replication. It is possible
in certain cell lines that virus replication could have occurred, but was
not detectable by visual observation on the phase or electron microscopes.
The development and use of shrimp baculovirus antisera for both polyhedrin
and infected tissue will enable continuation of our work with more confidence
and definitive applications. Immune peroxidase has been developed and is
now being routinely used for both insect baculovirus polyhedrins and en-
veloped nucleocapsid detection in this laboratory (Summers and Hsieh, manu-
script in preparation). It is known to be a reliable and semi-quantitative
technique. As soon as we obtain the shrimp virus antiserum, we will develop
a similar technology and application.
The serological relatedness of shrimp and insect baculovirus polyhedrins
is of considerable importance and the most positive aspect of this research
project. It demonstrates that insect and shrimp viral proteins do have a
similar antigen or antigenic determinants which are recognized by insect
baculovirus antisera. This has been confirmed in two kinds of serological
assays: immunodiffusion, which is good for specificity but weak in terms
of sensitivity, and radioimmunoassay (RIA), which is a very sensitive
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quantitative and specific assay. Both serological techniques are being
utilized for insect baculovirus detection and identification in this labora-
tory (Summers and Hoops, manuscript in preparation; Ohba et al., manuscript
in preparation). Furthermore, this technique has been shown to be highly
reliable. When shrimp virus antisera are available, we will attempt to
adapt RIA to that system as well. Therefore, if appropriate financial
support is available it will be possible to use three major serological
techniques: immunodiffusion, RIA, and immunoperoxidase. The complete
capability for shrimp baculovirus detection, identification, monitoring, and
screening will be available on both a quantitative and qualitative basis.
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SECTION 3
RECOMMENDATIONS
Attempts to develop _m_ v!tro systems for the replication and production
of shrimp baculovirus is highly desirable; however, it will require an em-
pirical approach and very likely will be a long-term effort. Many different
cell lines, conditions, and temperatures should be utilized in any attempt
to introduce the virus into an in vitro system. Furthermore, such studies
should not be conducted until a reliable screening procedure is available for
the detection of both polyhedrin production and enveloped nucleocapsid repli-
cation as discussed earlier. Baculoviruses are now known to replicate in the
absence of polyhedra formation and therefore the absence of the crystalline
structure does not ensure that virus replication is not occurring. It is not
practical to screen a variety of cell lines and/or experimental conditions
for virus replication by electron microscopy. The immunoperoxidase tech-
nique is easily and reliably adapted for this kind of screening procedure.
The study of serological relatedness of the shrimp baculovirus to insect
baculoviruses should be continued. With anticipated financial support, this
work will be continued in this laboratory as soon as the appropriate antisera
are available. Our inability to obtain a serological cross-reaction with
enveloped nucleocapsids is very likely due to the lack of sufficient concen-
trations of virus to detect such cross-reactions. More tissue and virus will
be required for such studies. Regardless, as stated earlier, the highest
priority research should be the development of RIA and immunoperoxidase
serological techniques in order to have available quantitative and qualita-
tive detection and monitoring technology.
Although academic at this time, continued studies of the shrimp DNA
should be encouraged, especially in the area of physical mapping of virus
genomes, by use of specific restriction endonuclease enzymes and hybridiza-
tion of apparently similar fragments.
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SECTION k
MATERIALS AND METHODS
SELECTION AND COLLECTION OF INFECTED ORGANISMS
Pink shrimp (^enaeus duorarum) were screened for tissues infected with
shrimp baculovirusT (P. duorarum NPV = PdS_NPV) at Gulf Breeze, Florida. The
availability of infected shrimp in large numbers was sporadic, and occasion-
ally natural dips occurred in the prevalence when infected material was not
available. The unavailability of shrimp on occasion was probably due to
several possibilities: 1) The shrimp obtained were not always from the
epicenter of the infected population, and fluctuations of infectivity from
the main source affected the availability; 2) The presence of an occasional
red tide in the general areas where the shrimp were obtained may have
resulted in the death of many patently infected organisms; 3) The shrimp
available perhaps were not from the area the supplier designated. Shrimp
were also selected from a fish market as an alternative to live shrimp;
that approach was not fruitful.
The data in Table 1 for the period of November 5 to December 5, 197^,
are representative of the magnitude of screening involved and the success
obtained in acquiring infected tissue for one shipment. All tissue was kept
at -90°C after dissection of the infected tissue from the organism and
shipped to Austin, Texas, in dry ice. All infected material was maintained
at ~90°C before and after virus purification.
PURIFICATION OF OCCLUDED PdS_NPV
NPV-infected shrimp hepatopancreatic tissue was homogenized in 0.2 M_
Tris-HCl containing 10"2 M_ EDTA, pH 7.8 (Tris buffer solution). The
homogenate was immediately centrifuged at 2,000 rpm at 5°C for two minutes
with the J-21 rotor and Beckman J-20 centrifuge. The pellet showed the
presence of a large number of characteristically occluded viruses as
visualized with brightfield and phase contrast light microscopy (Couch, 197^;
Couch et al., 1975). After low speed centrifugation, no visible polyhedra
remained in the supernatant. The supernatant was stored at -90°C, and the
2,000 rpm (480 x g) pellet resuspended by vortex mixing. One-half ml was
layered on linear sucrose gradients, ranging from ^0 to B0% (wt %}, and
centrifuged for 20 minutes at 33,000 rpm with the SW-^tl rotor. Most of the
pink color in the virus pellet remained at the top of the gradient. The
occluded virus banded at approximately one-half the distance in the gradient.
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TABLE 1. SCREENING AND COLLECTION OF INFECTED SHRIMP
FROM 11/5 TO 12/5, 1974
Date sample
completed
11/5
11/7
11/22
11/30
12/4
Total
Number shrimp
in sample
190
80
225
210
200
905
Number found
infected
25
2
0
0
0
27
°/
/o
infected
13.1
2.5
0
0
0
3.0
Shrimp type
Live
Live
Live
L i ve
Live
pinks
pinks
pinks
browns
pinks
Although purification was achieved by a single centrifugation, the
occluded virus band was removed from the sucrose gradient, pelleted by
differentia] centrifugation, and then rebanded on another linear 40 to
60% sucrose gradient. The occluded virus was removed for protein and
nucleic acid studies. The yield from a total of approximately 100 infected
hepatopancreatic tissues ranging from lightly to heavily infected was about
2.4 mg, as estimated from protein analysis.
SOLUBILIZATION OF OCCLUDED VIRUS
Attempts were made to solubilize occluded shrimp virus by using the
standard insect baculovirus technique. One-tenth ml of purified occluded
shrimp virus (170 yg) was pelleted and washed with deionized water. To the
pellet, dilute alkaline saline (DAS = 0.1 IM sodium carbonate plus 0.05 M
Nad, pH 10.9) was added. Occluded Autographa californica NPV (AcMNPV)~
was treated similarly. After two hr of exposure to DAS at room temperature,
no difference was observed in the light scattering of the shrimp baculovirus
solution, suggesting that solubi1ization had not occurred. Observation on
the phase microscope confirmed this. AcMNPV had been completely solubilized.
At the end of two hr exposure in DAS at 37°C, the shrimp virus preparation was
made 1% with respect to SDS. This was incubated for an additional one hr at
37°C when it was noted that no turbidity remained in the tube.
AM I NO ACID ANALYSIS
One hundred fifty ug of the PdSNPV purified by isopycnic banding in
sucrose gradients was pelleted, washed free of buffer solution, and resuspended
8
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in 0.2 ml of 1 mhl NH^CHOa (pH 11). The preparation did not immediately show
evidence of solubi1ization. Two-tenths ml of 15 M^ NH^OH was added. After
repeated heating at 70°C for two hr, solubi1ization was apparently achieved.
The solubilized protein was dried under vacuum, and samples were hydrolyzed
in aqueous HC1 at 105°C for 24 and 48 hr (Summers and Smith, 1976). Values
for threonine and serine were extrapolated to time zero.
SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
Electrophoresis in a 10.8% po1 vacrylamide vertical gel slab in the
presence of 0.1% SDS was done as described by Laemmli (1970). The samples
were prepared for electrophoresis by the addition of 2% SDS and 5% mercap-
toethanol in 0.25 M^ Tris (pH 6.8) with a final protein concentration of
1 mg/ml. The samples were heated at 100°C for three minutes and were sub-
jected to electrophoresis for three hr at a constant power of 7.5 watts/gel
slab. The gel slab was 14 x 12 cm and 1.5 mm thick. The gels were fixed
and stained with 25% isopropanol, 10% acetic acid, and 0.04% Coomassie
brilliant blue (R-250) for four hr and then destained in 10% acetic acid.
The method described by Weber and Osborn (1969) was used to determine
the apparent molecular weights of the viral proteins as compared to the
known weights of standard proteins.
PURIFICATION OF ENVELOPED NUCLEOCAPSIDS
Since it was not possible to recover enveloped nucleocapsids from
DAS-solubi1ized occluded virus, an attempt was made to recover the non-
occluded virus from the 2,000 rpm supernatant (see "Purfication of Occluded
Virus") of homogenized tissue. A modification of the purification procedure
for Oryctes NPV (Payne and Tinsley, 1974) was utilized. The 2,000 rpm super-
natant in Tris buffer solution was centrifuged at 50,000 x g for 60 min with
the SW-27 rotor. The supernatant was immediately stored at -90°C. One-half
ml of the Tris buffer solution was added to the pellet and then allowed to
set in the refrigerator overnight. The pellet was resuspended by vortex
stirring and layered on top of a 25-50% (wt %) sucrose gradient constituted
in the Tris buffer. The gradients were centrifuged at 100,000 x g for 90 min
with the SW-41 rotor. The gradients also contained 1 ml of a 60% (wt %)
sucrose cushion.
CELL CULTURE STUDIES
Homogenized Pd^NPV infected hepatopancreatic tissue was added to each of
the following cell lines in order to investigate ability to infect inverte-
brate cells: Trichoplusia ni (TN-368), Spodoptera frugiperda, Aedes
a I bop ictus, Culex salinarius, Armigeres subalbatus. The cell lines were main-
tained at 22°C and 28°C post infect ion to see if temperature influenced suscep-
tibility or replication.
DNA EXTRACTION
Occluded shrimp baculovirus was purified by equilibrium banding on sucrose
?radients as described previously. Since it had been demonstrate^ that enve-
oped nucleocapsids could not be separated from the crystal protein by routine
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DAS solubi1ization techniques, intact shrimp polyhedra were adjusted to a
final concentration of 2% with respect to sodium lauryl-sarcosine in 1 x SSC
(sodium saline citrate; 0.015 M sodium citrate, 0.15 M. NaCl), 10 mM^ EDTA, pH
7.0 (Summers and Anderson, 1973"). The sarcosyl treated polyhedral prepara-
tion was then heated at 60°C for 30 min. The solution cleared and was layered
on top of a continuous gradient (density range 1.4 to 1.6 g/ml) ethidium
bromide-cesium chloride (Summers and Anderson, 1973). Centrifugation was
conducted for 2k hr at 40,000 rpm with the SW-65 rctor. After centr if'ugct ion,
the gradients were visualized and photographed under ultraviolet light and
the DNA bands recovered for Kleinschmidt preparations as described previously
(Summers and Anderson, 1973; Summers, 1977).
DNA SPREADING AND ELECTRON MICROSCOPY
The Kleinschmidt spreading technique described by Lee, Davis, and
Davison (1970) was utilized to visualize the circular forms of PdSNPV as
well as estimate size. For estimates of size by measurements relative to
calibrated standards, the Hitachi HU-11E electron microscope was used with
a Fullam grid standard calibrated at 28,800 lines per inch. Also the
densit^ of DNA relative to DNA standards was estimated to be approximately
2 x 10 daltons per micron relative to purified T7 DNA bacteriphage standards.
IMMUNODIFFUSION
Double immunodiffusion was performed by a modification of the methods
described by Oucterlony (1962). Briefly, 8 ml of \% agarose in 0.01 M
Tris buffer, pH 7-8, containing 0.01 M_ EDTA and 0.01% sodium azide was
poured into disposable immunodiffusion plates (Miles Laboratories, Elkhart,
IN), and wells 3 mm in diameter were cut around a 4-mm central well with
a 4 mm centeito-center distance. The central well was filled with 20 yl
of the appropriate undiluted polyhedrin antiserum. The peripheral wells
were then filled with 10 yl of a 1 yg/yl solution of each antigen to be
tested. The plates were placed in a humid atmosphere and incubated for
2k to 48 hr.
COMPETITION RADIOIMMUNOASSAY (RIA)
A modification of the micro solid-phase radioimmunoassay (RIA) tech-
niques described by Purcell et al. (1973) was employed. Briefly, wells of
a polystyrene microtiter plate (Limbro Scientific Co., New Haven, CT) were
coated with 200 yl of a 0.02 V(_ NaHCO buffer, pH 9.6, containing 25 yg/ml
of Protein A (Pharmacia Fine Chemicals, Piscataway, NJ) and incubated for
18 hr at 4°C. The wells were aspirated and washed twice with 200 yl of
0.01 M^ Tris buffer, pH 7-8, containing M^ EDTA, 0.005 M^ Kl , and 0.01%
sodium azide. Two hundred ml of a 1:500 dilution of AcMNPV polyhedrin
antiserum in Tris buffer was added to each well and incubated for 18 hr
at 4°C. The wells were then aspirated, washed twice with Tris buffer, and
coated with 200 yl of Tris buffer containing 5% BSA to prevent non-specific
adsorption of antigens to unreacted sites on the wells. Following incuba-
tion for 4 hr at 4°C, the wells were washed twice with Tris buffer, and
10-fold concentrations of polyhedrins or granulins (0.01 to 1000 ng) were
added and incubated for 18 hr at 4°C. The wells were then aspirated and
10
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washed three times with Tris buffer. One nanogram of 1251-AMN polyhedrin
was then added to each well and incubated an additional 18 hr at ^°C. The
wells were aspirated, washed three times with 5% BSA-Tris buffer, cut out,
and counted for 1 min each in a gamma spectrophotometer (Searle, Model 1195,
Arlington Heights, IL).
11
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SECTION 5
RESULTS AND DISCUSSION
PURIFICATION OF POLYHEDRA
After banding in sucrose gradients, a very sharp light scattering zone
was observed (Figure 1). Observations of a sample of the zone on the phase
microscope revealed the presence of occluded virus.
The Tris-buffering solution did not appear to be a satisfactory or
adequate medium in which to purify and maintain the occluded shrimp NPV.
Periodic observations demonstrated that the crystals underwent fragmenta-
tion and/or disruption. Therefore, it was necessary to work quickly during
the homogenizing and extraction procedure to obtain an optimal yield of
occluded virus.
Subsequent storage of the purified occluded virus preparation also did
not seem to stabilize the breakdown of the polyhedra. After a year of
storage at -20°C, very few intact polyhedra could be observed by phase
microscopy, even though the total protein concentration remained constant.
The addition of 0.1% SDS to the shrimp baculovirus in Tris buffer
results in immediate solubi1ization of the polyhedra. A similar effect
occurs by the addition of NP-40 at a final concentration of 1.0%. Unfor-
tunately, enveloped nucleocapsids were not recovered from the preparation
after this treatment.
The process of polyhedra dissociation as observed by phase microscopy
is significantly different from that for AcMNPV. This was visualized by
placing the baculovirus preparation on glass slides with a cover slip.
One-tenth N^ NaOH was applied to the edge of the cover slide, and the sequen-
tial solubi1ization of the polyhedra was easily observed as the basic
solution diffused beneath the cover slip. With AcM_NPV, polyhedra solubilized
immediately after exposure to the NaOH solution. The refract!le polyhedra
were observed to change to a dark color. However, the polyhedral "membrane"
remained intact. The Brownian movement of bundles of enveloped nucleocap-
sids could be clearly observed inside the intact polyhedral membranes at
high magnification on the phase microscope. In comparison, the shrimp
baculovirus crystal began to fragment into smaller units, each of the
smaller fragments undergoing subsequent breakdown until apparent solubili-
zation occurred. This technique demonstrated that no polyhedral membrane
could be observed in association with shrimp polyhedra. The presence of
the polyhedral membrane associated with insect baculovfruses and the absence
of such a structure on the shrimp NPV have been confirmed by electron
12
-------
Figure 1. Isopycnic banding of shrimp baculovirus polyhedra in sucrose
gradient. Density range: 1.1-1.3 g/ml. Conditions and details of the
procedure are specified in "Materials and Methods."
13
-------
microscopy (Summers and Arnott, 1969; Harrap, 1972; Couch, 197*0-
For these studies it was not possible to isolate intact enveloped
nucleocapsids from shrimp virus polyhedra; the routine DAS solubi1ization
did not work and the use of ionic and nonionic detergents disrupted the
physical integrity of the virus particle. Exposing the shrimp virus poly-
hedra to higher pH conditions may be effective. However, sufficient
quantities of occluded virus were not available for continued experiments
based on this approach. It was decided that serological studies would have
higher priority and that continued studies attempting to isolate the en-
veloped nucleocapsids could be conducted later if enough material was
available. This subject will be dealt with in the section on our attempts
to extract enveloped nucleocapsids directly from infected hepatopancreatic
ti ssue.
PURIFICATION OF ENVELOPED NUCLEOCAPSIDS FROM INFECTED TISSUE
As represented in Figure 2, four major zones were fractionated in su-
crose gradients after centrifugation of the infected tissue homogenate.
The top of the gradient where the sample was layered contained all of the
pink color characteristic of the 50,000 x g pellet. The major band in the
lower one-third to three-fourths region of the gradient was removed and
only some unidentified material was observed by phase and electron micro-
scopy.' All four of these fractions were carefully removed by syringe, cen-
trifuged at 50,000 x g for 60 min, and the pellets suspended in Tris buffer
for observation on the electron microscope.
Enveloped nucleocapsids were observed only in the intermediate region,
which showed some slight opaque or light refractile material but which did
not exhibit any distinct bands. Since very few enveloped nucleocapsids were
observed in this sample, it was not considered feasible to attempt to purify
and recover enveloped nucleocapsids directly from homogenized tissue. Con-
siderable quantities of tissue would be required and very likely are not
available.
SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
Shrimp polyhedrin prepared and maintained properly to minimize break-
down of the protein during storage and handling shows the presence of only
one major band (Figure 3) with a molecular weight of 50,000. Polyhedrir
handled frequently and at room temperature degrades. The molecular weight
of 50,000 is distinctly different and much larger than that for insect
baculovirus granulins and polyhecrins (Figure 3)- Although Summers and
Smith (1976) demonstrcted that granulins and polyhedrins were approximately
28,000 daltons, the results reported here show that there is some varia-
bility in molecular weights of individual polyhedrins as measured by the
improved SDS-PAGE technique. Of the granulins and polyhedrins shown, the
molecular weights range from 25,500 to 31>000 daltons."
The size difference as measured by SDS-PAGE can be correlated with
observations by electron microscopy (Summers and Arnott, 1971; Harrap,
1972; Couch, 197*0- The crystal lattice spacing for most NPVs and GVs has
-------
HOMOGENIZED SHRIMP
HEPATOPANCREAS
480 x g
PELLET .
OCCLUDED
VIRUS
.SUPERNATANT
PELLET-
5,000 x g, 15 min.
SUPERNATANT
5,000 x g, 60 min.
DISCARDED
PELLET
LAYERED ON 25-50%
CONTINUOUS SUCROSE
GRADIENT
110,000 x g, 90 min.
SAMPLE
INTERMEDIATE ZONE
MAJOR BAND
CUSHION INTERFACE
SUPERNATANT
DISCARDED
Figure 2
Figure 2. Purification schematic for fractionation of infected shrimp hepato-
pancreatic tissue.
15
-------
A BC DEFGHIJ KL
W» 150,000
90,000
*» 68.00O
«*. 53,000
40.000
31,000
- - 25,000
17,200
t t t t I f " t t t t
£8sl8 £828 £828 58 58
zozo zozo zozo cm - o
' -
Figure 3- SDS-polyacrylamide gel electrophoresis of shrimp polyhedrin rela-
tive to insect baculovirus polyhedrins.
16
-------
been measured to range from 40 to 70 Angstroms (A). The subunit comprising
the shrimp baculovirus crystal appears to be distinctly larger since the
lattice space is approximately 120 A.
AM I NO ACID ANALYSIS
Amino acid analyses show (Table 2) that shrimp polyhedrin has approxi-
mately 471 residues based upon a molecular weight of 50,000 as determined
by SDS-PAGE.
A comparison of the data in the table shows that insect baculoviruses
exhibit remarkable similarity of amino acid composition. Although the
shrimp polyhedrin also exhibits some general similar relationship, the
shrimp polyhedrin does show distinct differences for certain amino acids:
phenylalanine, tyrosine, isoleucine, methionine, glycine, and serine.
Although in general amino acid analyses are usually not definitive as in-
dicators of specific differences between or among proteins, data suggest
some marked differences in amino acid sequences between the shrimp poly-
hedrin and those from insect baculoviruses. Consequently, it is not possi-
ble to discuss specific similarities or dissimilarities of the polyhedrins.
CELL CULTURE STUDIES
Although not given major emphasis at this time, preliminary attempts
were made to infect invertebrate cell lines with homogenized heavily infected
hepatopancreatic tissue.
Only Sppdoptera frugiperda cells showed a cytopathic effect (CPE).
Limited electron microscope observations of exposed S^. frugiperda eel 1 s did
not reveal any virus replication.
That study did not confirm whether the observed CPE was a result of
some toxic effect of shrimp tissue or some effect of the virus. In an
attempt to resolve this question, fractions from the gradient of fractionated
viral material discussed previously were utilized for CPE studies. Spodop-
tera frugiperda cells were exposed to material from top, intermediate, major
band, and cushion interface fractions. All preparations induced a CPE after
three days' exposure. These observations indicated that the CPE was not a
result of virus activity since apparently uninfected shrimp hepatopancreatic
tissue also produced a similar CPE.
The CPE had the following characteristics: the cells exhibited some
nuclear hypertrophy as well as the development of some refract!le bodies in
the cell. It was first thought that the refractile bodies were nuclear in
origin. However, it is now believed that they were cytoplasmic after ob-
servations of the cells on the electron microscope. The size of the
.§. f rug iperda_ eel 1 s made it difficult to differentiate the position of the
refractile bodies in the cells. However, because of their size, refractile
nature, and apparent position in the cells, it was easy to mistake the
refractile bodies for proteinic crystals characteristic of insect baculovirus
infections.
17
-------
TABLE 2. AM I NO ACID ANALYSIS OF GRANULINS AND POLYHEDRINS
00
Ami no acid
Asx
Thr
Ser
Glx
Pro
Gly
Ala
Val
l/2Cys
Met
lieu
Leu
Tyr
Phe
Lys
His
Trp '
Arg
Total
Molecular
TnGV
10.5
6.3
4.5
11.8
6.6
5-3
5.4
7-0
1.7
0.4
6.1
8.2
3.2
6.7
5.5
3-4
3.2
6.0
weight
SfGV
10.3
4.4
4.8
13.6
6.8
6.3
5.0
5-7
1.5
0.5
5-7
8.3
4.1
4.1
6.6
2.5
3-7
. 6.3
Mole
AcMNPV
10.8
4.1
2.9
11.1
7-1
5-5
5.6
8.1
2.2
0.4
5.3
7-0
4.9
5.2
8.6
2.3
2.9
6.1
*
TnSNPV
11.4
4.4
4.4
12.3
7-5
7.0
6.0
7-6
1.4
0.4
5-0
7.8
4.0
3.7
8.2
2.3
2.0
5-0
RoMNPV
11.6
3.8
3.8
12.3
6.7
5.8
3.8
8.2
1.3
0.3
6.0
6.7
5-0
4.9
8.8
2.4
2.5
6.0
PdS_NPV
Res idues
TnGV* SfGV*
12.3 23.6
5.6
14.3
8.3 10.1
10.8
26.6
5.4 14.8
11.0
12.0
6.4 12.2
7.5
15.7
2.1 3.9
1.5
0.91
3-9 13.8
6.9
18.6
0.2 7-2
1 .2
15.2
8.9 12.4
3-3
7-7
3-3
5.0
13.5
225.8
25,500
23.4
10.0
11 .0
30.9
15.3
13.8
11.4
12.9
3.3
1.2
12.9
18.8
9.4
9.2
15.0
5-7
9.2
14.3
227.7
26,000
AcMNPV*TnSNPV*RoMNPV PdSNPV
28.4
10.7
7-7
29.0
18.4
14.5
14.9
21.1
5-9
1.1
14.0
18.3
12.8
13-7
22.4
6.1
7-5
15-9
262.4
30,000
31.7
12.3
12.3
34.1
20.8
19.4
16.6
21.1
4.0
1.1
14.1
21.7
11.2
10.4
22.8
6.3
5.5
13.8
279.2
31 ,000
30.1
9-9
9.9
32.2
17.6
15.2
10.0
21.4
3-3
0.6
15.8
17-6
13.1
12.8
22.8
6.2
6.5
15.8
260.9
30,000
58
26
39
50
26
51
30
35
9
7
19
33
1
5
42
16
24
471
sn.ooo
* Summers and
Smith,
1975
-------
In order to conduct a more elaborate preliminary study the following
experiment was conducted with TN-3&8-10, Culex tritaeniorhynchus, Spodop-
tera frugiperda, Armjgeres subalbatus, and Culex salinarius eel 1s. Each
cell line was maintained at both 22°C and 28°C and exposed to homogenized
infected hepatopancreatic tissue. In contrast to the first exposure period
of only A8 hr, cells were prepared for electron microscopy after 2 and k days,
An extensive and comprehensive search of infected cells by electron
microscopy was not conducted; however, preliminary observations of selected
samples from those exposed cell lines did not show the presence of replica-
ting virus. However, compared to the controls, exposed cells showed some
distinct alteration of nuclear and cytoplasmic ultrastructure and some nu-
clear hypertrophy, as well as chromatin margination. It is difficult to
evaluate the extent of nuclear membrane proliferation, although it did
appear in a few observations. Since observations were made primarily for
the purpose of detecting replicating virus, the differences between control
and shrimp virus infected cells cannot be discussed here without a more
extensive investigation.
PdSNPV DMA
Ethidium bromide-cesium chloride gradients containing shrimp baculovirus
DMA exhibited the typical banding profile characteristic of a mixture of
covalently closed DNA (ccDNA) and relaxed circular DNA (rcDNA) and double-
stranded linear (dlDNA) molecules (Figure k) (Summers and Anderson, 1973)-
The positions of the bands were not accurately measured, but with respect
to the density of TA DNA and the results of previous studies on insect
baculovirus DMAs, the shrimp virus ccDNA banded at the characteristic density
of 1.58 g/ml and the band characteristic of dlDNA and rcDNA banded at approx-
imately 1.5^ g/ml. The intensity of the bands under illumination by UV
light revealed that the relative proportions of ccDNA at 1.58 g/ml to that
of dlDNA and rcDNA at 1.5** g/ml was very similar to that of insect baculo-
virus DNA. Therefore, the yield of ccDNA was approximately 20 to 30% that
of the total DNA preparation.
Since only limited quantities of the shrimp PdjJNPV DNA were available
(for example, the total amount in Figure k represents approximately 3 to 5
yg DNA), sedimentation studies for estimates of molecular weights were not
feasible. Therefore, purified viral DNA recovered from the gradients
shown in Figure 4 were prepared (Lee et al., 1970) for observation on the
electron microscope.
The average mean molecular weight determined from several measurements
on ten different molecules was 75 x 106 daltons ± 2 x 106. As can be seen
from Figures 5~10 the characteristic conformation of a rcDNA is apparent.
Figure 1 appears to be supercoiled DNA.
In a recent review on baculovirus DNAs (Summers, 1977), it has been
shown (Table 3) that baculovirus DNAs range in size from 75 to 100 million
daltons. Recent unpublished information (personal communication with J.
Longworth) suggests that direct measurements on baculovirus genomes are
going to be 10 to 2Q% less than molecular weights estimated by hydrodynamic
19
-------
4A
4B
Figure 4. Equilibrium banding of shrimp baculovirus DNA in ethidium bromide-
cesium chloride gradients. Methods and procedures according to Summers and
Anderson (1973). Arrow designates covalently closed DNA (1.58 g/ml).
Figure 4A. T, bacteriophage DNA. Figure 4B. Shrimp baculovirus DNA.
20
-------
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Figure 5. Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 29.4 mm
21
-------
^S£$I^^5^
Figure 6. Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 15.8 mm
22
-------
Figure 7. Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 89.3 mm
23
-------
:
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;,^ -: .-.*' 'S'n-'-v
*? . .. «, " . ' i, : ^ > ' ' l -' '
,*.'.- .;.'^:'...'- -:.
* » _--'
Figure 8. Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 16.2 mm
:«;-::.;
. . -
.- - ' «
-------
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Figure 9- Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 16.6 mm
-------
lx£\££:vv£-.-.V ^:'':s'-:^'.-^:^:y~<.s^.\\:
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Figure 10. Kleinschmidt preparation of shrimp baculovirus DNA.
1 micron = 15.4 mm
26
-------
TABLE 3. DEOXYRIBONUCLEIC ACIDS OF BACULOVI RUSES*
M
V i rus
Agrotis segetum NPV
Aporia crataegi NPV
Autographa californica NPV
Bombyx mori NPV
Bombyx mori NPV
Bombyx mori NPV
Botnbyx_ mori NPV
Bombyx mori NPV
Bombyx mori NPV
Hel iothi s artnigera
Size (x 106) Sw,20
30
-Q
i>o
Q£ 1 1 Q _____
ofa- 1 I o
76
2.0 13- IS
up to 10 1^.5^
2k and l»8t 351, ^i
loot
59-ll8t l^OS, 9^S, 61S, 45S, ]ks
50
Reference
(2*)
(1)
McCarthy (personal
communication)
CD
C20)
(5)
(30)
(17)
(18)
(22)
"Table from Summers, 1977
tPresence of circular DNA molecules
(continued)
-------
M
CO
TABLE 3 (continued)
Virus
Lymantria dispar MNPV
Oryctes rhinoceros NPV
Penaeus duorarum SNPV
Rachiplusia ou MNPV
Spodoptera frugiperda MNPV
Trichoplusia ni SNPV
Dendrol imus sibirlcus GV
Heliothis armigera GV
Spodoptera f rug i per da
Trichopjusia ni GV
Size (x 106)
56
87*
75*
91*
95*
95*
80*
50*
95*
100*
sw,20
57. 2£
581
591
591
591
60S
Reference
(0
(21)
Summers and Couch
(unpubl ished)
(27)
(27)
(27)
(23)
(22)
(25) (27)
(25) (26)
*Presence of circular DNA molecules
(continued)
-------
TABLE 3 (continued)
N>
V i rus
Aporia crataegi NPV
Bombyx mori NPV
Bombyx mori NPV
Bombyx mori NPV
Heliothis armigera NPV
Hemerocamp^ pseudotsugata NPV
Lymantria dispar NPV
Oryctes rhinoceros NPV
Rachipjusia ou MNPV
Spodoptera f rugjpenda MNPV
Spodoptera littoral is NPV
Hel iothis armigera GV
Spodoptera frugiperda GV
Trichoplusia ni GV
Tm (host)*
86-87
87.5
71t
71. 5t
72. 8t
87-5 (83.5)
72t
74. 5t
69. 3t
G&C (%)
41.9
42.7
43
43
46
50
37.5
Density (host)-'- % DNA Reference
/ 1 Q \
i (L f /i \
21
(l\
1.70 (1.9)
22
1.6994 (25) (26)
"Density of host DNA is placed in parenthesis for comparison with viral DNA.
tDetermination made in 0.1 x SSC.
-------
techniques utilizing sedimentation velocity. With this in mind, and with the
molecular weight estimate derived herein, it is very likely that the shrimp
baculovirus DNA falls very nicely into the size range of typical insect bacu-
lovirus DMAs.
Because of the limited amount of baculovirus DNA available for these
studies, it was not possible to explore additional chemical and physical
properties of the shrimp virus DNA. However, recent advances in molecular
biology involving in vitro labeling of DNA combined with restriction endo-
nuclease or physical mapping now may allow for comparisons between shrimp
baculovirus DNAs and insect virus DNAs.
SEROLOGY
Immunod iffus ion Assays
It has been determined in this research that PdSJNPV polyhedrin is
extremely insoluble to the routine DAS solubi1ization procedure. Conse-
quently, initial attempts to reveal relatedness of shrimp NPV and insect NPV
polyhedrins by immunodiffusion were inconclusive and in general demonstrated
no cross-reaction. Therefore the use of detergent was necessary for a more
complete disaggregation or solubi1ization of the shrimp NPV polyhedrin and
experiments were conducted in the presence of various concentrations of SDS
in an attempt to reveal any similar antigenic determinants in the shrimp
polyhedrin. The results in Figure 11 show that after treatment with 0.1%
SDS shrimp polyhedrin reacts against AcMNPV polyhedrin antisera. In Figure
11 the effect of the SDS treatment also unmasks a reaction with TnGV granulin
antisera but no reaction with 1% NP-^0 treated or untreated shrimp polyhe-
drin. A control (not shown) was conducted wherein the homologous antigen
and antiserum were tested in the presence of 0.1% SDS. This did not reveal
any artifacts.
These cross-reactions correlate with immunodiffusion analyses of AcMNPV
and TnGV antisera. Both insect baculovirus antigens and antisera cross-
react showing a strong partial identity (Summers and Hoops, unpublished
data). Therefore, one would expect that related sequences present in
shrimp polyhedrin would also react with the two insect baculovirus polyhedra
antisera. This reaction was confirmed.
The control studies and conditions discussed in figure legends showed
that 0.1% SDS had no deleterious effects on the antigen-antibody reaction,
although the precipitant bands appeared slightly more diffuse than those
occurring with the untreated proteins. Furthermore, preimmune sera did not
react with any of the SDS-treated polyhedrins or granulins tested. Also,
the 1.0% NP-AO treatment did not facilitate any reaction in the immuno-
diffusion assay.
Immunodiffusion assays were also performed with fractionated .infected
shrimp hepatopancreatic tissue. The pellets obtained by differential cen-
trifugation of the tissue at 8,000 and 100,000 x g and the supernatant of
the 100,000 x g spin were tested against AcMNPV polyhedrin, virus, and TnGV
granulin antisera. All samples were again treated with 0.1% SDS prior to
30
-------
Figure 11. Immunodiffusion of shrimp polyhedrin. Figure 11A. Center well,
AcMNPV polyhedrin antiserum; well 1, untreated shrimp polyhedrin;.wel1s 1, 3,
and 5, AcMNPV polyhedrin; welIs 2, k, and 6, 0.1% SDS-treated shrimp polyhe-
drin. Figure 11B. Center well, TnGV granulin antiserum; wells 1, 3, and 5»
TnGV granulin; well 6, 0.1% SDS-treated shrimp polyhedrin; well 2, \% NP-^0
treated shrimp polyhedrin; well k, untreated shrimp polyhedrin.
Figure- 12. Immunodiffusion .of infected shrimp hepatopancreatic tissue.
Figure 12A. AcMNPV polyhedrin antiserum, center well; wells 2, A, and 6,
AcMNPV polyhedrin; wel1 1, 8000 x g infected shrimp hepatopancreatic tissue
pellet; well 3, 8000 x g shrimp supernatant; well 5, 100,000 x g shrimp tissue
pellet. Figure 12B. TnGV, center well; wells 2, k, and 6, TnGV granulin;
well 1, 8000 x g infected shrimp hepatopancreatic tissue pellet; well 3,
8000 x g shrimp supernatant; well 5, 100,000 x g shrimp tissue pellet.
3.1
-------
assay. Cross reactions with both AcMNPV polyhedrin and TnGV granulin are
observed in Figures 12 A-B. AcMNPV polyhedrin appears to react with all
three fractions of the infected hepatopancreatic tissue whereas TnGV reacts
only with the 8,000 x g pellet. The reasons for these differences are
unclear at present.
The fractionation protocol for infected shrimp tissue (Figure 2) was
intended primarily to concentrate nonoccluded Pdj>_NPV virions for a compari-
son with AcMNPV enveloped nucleocapsid antisera. The reactions not shown
here were all negative, implying two possibilities: 1) there was no sero-
logical relationship between or among viral antigenic determinants, or 2)
the concentration of virus protein in the various fractionations was not
adequate to reveal any cross-reactions. Because of the latter possibility,
further studies are needed with greater quantities of shrimp hepatopan-
creatic tissue before more definitive conclusions can be made. Although
not available at present, antisera are being prepared against shrimp baculo-
virus polyhedrin and infected hepatopancreatic tissue. Once the antisera
have been collected and tested for titer, a complete comparison of the
shrimp baculovirus system with insect baculovirus antigens will be rerun.
Since the insect baculovirus polyhedrins for AcMNPV and TnGV possess
common primary sequences (Summers and Smith, 1976), the cross reactions with
those two antisera reveal that relatedness in the primary structure between
the two proteins does exist and is detected by the immunodiffusion assay.
Therefore, cross reactions obtained with PdS_NPV polyhedrin against both of
the insect baculovirus antisera indicate that the shrimp polyhedrin contains
some similar primary sequence(s) and therefore common or similar antigenic
determinant(s) to those found in insect baculovirus polyhedrins and granu-
lins. However, a careful analysis of the results also suggests that PdS_NPV
polyhedrin may contain different antigenic determinants. Further studies
are needed to clarify these relationships.
Rad i o ? mmunoa s say _(_RIA)
Figure 13 shows the results of competition RIA of shrimp polyhedrin
versus AcMNPV polyhedrin and its homologous antiserum. The results show that
if shrimp polyhedrin is not treated with SDS in order to denature the pro-
tein and expose reactive sites, it does not compete in the RIA. However,
if treated with 0.1% SDS, it does compete but at a level 5,000-fold greater
than the homologoud system. This confirms that some related sequence is
available in the shrimp polyhedrin protein. This technique is quantitative,
reproducible, and sensitive. The comparisons with other insect NPVs are
utilized to reveal the degree of relatedness of the shrimp polyhedrin. As
can be observed, shrimp polyhedrin is the least related of all the baculo-
virus polyhedrins investigated to date.
32
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100
HoMNPV
o
QQ
PdSNPV
2 I 01234
Nanograms Competing Antigen (log)
Figure 13. Analysis of shrimp polyhedrin by competition radioimmunoassay
with anti-AcMNPV polyhedrin. The assay employed 1 ng of 125I-AcMNPV poly-
hedrin (80,000 cpm/ng) and anti-AcMNPV polyhedrin serum (1:2500 final dilu-
tion). The competing proteins, purified AcMNPV polyhedrin (), HaMNPV
G^), SfGV granulin ( ), and shrimp polyhedrin (^^ ), were added in
increasing 10-fold concentrations as indicated.
33
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REFERENCES
1. Bonami , J. R. Viruses from Crustaceans and Annelids: Our State of
Knowledge. In: Proc. Fourth Int, Colloq. Invertebrate Pathol.,
Queen's University, Kingston, Ontario, Canada, 19/6. pp. 20-23.
2. Couch, J. A. An Enzootic Nuclear Polyhedrosis Virus of Pink Shrimp:
111 trastructure, Prevalence, and Enhancement. J. Invertebrate Pathol.
24:311-331,
3- Couch, J. A. 1976. Attempts to Increase Baculovirus Prevalence in
Shrimp by Chemical Exposure. Prog. Exp. Tumor Res. 20:304-314 (Karger,
Basel).
4. Couch, J. A., M. D. Summers, and L. Courtney. Environmental Signifi-
cance of Baculovirus Infection in Estaurine and Marine Shrimp. Ann.
N.Y. Acad. Sci. 266:528-536, 1975-
5. Couch, J. A. and L. Courtney. Interaction of Chemical Pollutants and
Virus in a Crustacean: A Novel Bioassay System. Ann. N.Y. Acad. Sci.,
1977 (in press).
6. Harrap, K. A. The Structure of Nuclear Polyhedrosis Viruses. II. The
Virus Particle. Virology 50:124-132, 1972.
7- Johnson, P. T. A Baculovirus from the Blue Crab, Calllnectes sapidus.
In: Proc. Fourth Int. Colloq. Invertebrate Pathol., Queen's University,
Kingston, Ontario, Canada, 1976. p. 24.
8. Laemml i , J. K. Cleavage of Structural Proteins during the Assembly of
the Head of Bacteriophage T4. Nature 227=680-685, 1970.
9. Lee, C. S., R. W. Davis, and N. Davidson. A Physical Study by Electron
Microscopy of Terminally Repetitious, Circularly Permuted DNA from
Coliphage Particles of Escherichia col i B. J. Mol . Biol . 48:1-22, 1970.
10. Oucterlony, 0. Diffusion-in-Gel Methods for I mmuno logical Analysis.
In: Progress in Allergy, Vol VI., P. Kallos and B. H. Woksman, eds.,
New York, Kayor, 1962. pp. 30-154.
Payne, C. C. The Isolation and Characterization of a Virus from
Oryctes rhinoceros. J. gen. Virol. 25:105-116, 1974.
Payne, C. C. and T. W. Tinsley. Structural Proteins and RNA Components
of a Cytoplasmic Polyhedrosis-Vi rus from Nymphal is io (Lepidoptera:
34
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Nymphalidae). J. Gen. Virol. 25:291-302, 1974.
13- Purcell, R. H., D. C. Wong, H. J. Alter, and P. V. Holland. Microtiter
Solid Phase Radioimmunoassay for Hepatitis B Antigen. App]. Microbiol.
26:478, 1970.
14. Summers, M. D. 1977- Deoxyribonucleic Acids of Baculoviruses. In:
Viruses in Agriculture, Beltsville Symposium, May, 1976. pp. 233~246.
15. Summers, M. D. and D. L. Anderson. Characterization of Nuclear Poly-
hedrosis Virus DNAs. J. Virol. 12:1336-1346, 1973-
16. Summers, M. D. and G. E. Smith. Comparative Studies of Baculovirus
Granulins and Polyhedrins. Intervirology 6:168-180, 1976.
17- Summers, M. D. and H. J. Arnott. Ultrastructural Study on Inclusion
Formation and Virus Occlusion in Nuclear Polyhedrosis and Granulosis
Virus-Infected Cells of Trichoplusia ni. J. Ultrastruct. Res. 28:462-
480, 1969.
18. Volkman, L. E., M. D. Summers, and C. Hsieh. Occluded and Nonoccluded
Nuclear Polyhedrosis Virus Grown in Tnchoplusia ni: Comparative
Neutralization, Comparative Infectivity and _m_ vj_t£o_ Growth Studies.
J. Virol. 19:820-832, 1976.
19. Weber, K. and M. Osborn. The Reliability of Molecular Weight Deter-
mination by Dodecylsulfate-Polyacrylamide Gel Electrophoresis. J. Biol.
Chem. 244:4406-4412, 1969-
20. Wildy, P. Classification and Nomenclature of Viruses. Monographs in
Virology 5:17-19, 197K
« U.S. OOVEENME.VT PRINTING OFFICE: 1978 74O-263/1S24 Region No. 4
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-77-13Q
2.
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
Characterization of Shrimp Baculovirus
5. REPORT DATE
October 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Max D. Summers
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Cell Research Institute
University of Texas
Austin, Texas 78712
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
EPA Grant R-803395
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
U.S. Environmental Protection Agency
Sabine Island
Gulf Breeze. Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/0^
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The research undertaken involved the partial characterization of a baculovirus of
the pink shrimp, Penaeus duorarum. The significance of the study is related to the fac
that the shrimp baculovirus is morphologically similar to insect vaculoviruses which
were considered unique to insect arthropods prior to the discovery of shrimp nuclear
polyhedrosis baculovirus (NPV). Further, insect baculoviruses are being developed and
applied as microbial pesticides for the control of certain agricultural insect pests.
Whereas the baculovirus diseases in pests of agricultural or medical importance are
considered a desirable relationship, a baculovirus infection in shrimp is an undesirabl
one.
Research included investigations of the biochemical, structural, and, where
appropriate, biological properties of the shrimp virus as compared to those of known
and characterized properties of insect baculoviruses, both granulosis and NPVs.
Evidence for any structural relatedness of the shrimp NPV to insect NPVs has been
confirmed in cross-reactions of purified shrimp NPV polyhedrin and infected shrimp
tissues to insect baculovirus antisera.
This report covers the period September 23, 197*+ to December 31, 1976.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTlFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Arthropod virus
Shrimp Baculovirus
physical and chemical characterization
Biological Control Agents
vi ruses
Shrimp Baculovirus charac
Biolgoical Control Agents
Natural Pathogens
teristics
and
3. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report/
unclassified
21. NO. OF PAGES
36
20. SECURITY CLASS (This page)
unclassi fied
22. PRICE
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