Biochemical characterization and time-course analysis of Lymantria dispar nuclear polyhedrosis virus with monoclonal antibodies1 CAO-Guo y u 2 Department of Entomology, University of Maryland, College Park, MD 20742, U.S. A . Department of Entomology and Center of Agriculture Biotechnology of the Maryland Biotechnology Institute, University of Maryland, College Park, MD 20742, U.S.A.

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TSUEYDING Institute of Zoology, Academia Sinica, Beijing, People's Republic of China FRANKHETRICK Department of Microbiology, University of Maryland, College Park, MD 20742, U.S. A . AND

HEI-TI H s u U.S. Department of Agriculture, Agriculture Research Service, Florist and Nursery Crops Laboratory, Beltsville, MD 20 705, U.S. A . Received July 2, 1991 Revision received October 8, 1991 Accepted October 15, 1991 Yu, C.-G., MA, M., DING,T., HETRICK,F., and Hsu, H.-T. 1992. Biochemical characterization and time-course analysis of Lymantria dispar nuclear polyhedrosis virus with monoclonal antibodies. Can. J . Microbiol. 38: 248-257. Hybridoma cell lines secreting monoclonal antibodies (MAbs) specific to a 31 000 molecular weight viral protein or a 3 1 000 molecular weight polyhedrin protein of Lymantria dispar nuclear polyhedrosis virus (LdNPV) were developed. The two polypeptides were shown to be different by comparing their amino acid compositions. Immuno-electron microscopy was used to verify specific binding of the MAbs to their respective targets. Specific MAbs were used to develop an ELISA procedure to monitor the development of LdNPV virus and polyhedrin in vivo. Results indicated that in hemolymph of larvae fed lo6 polyhedral inclusion bodies, the concentration of virus began to increase 16 h after inoculation and continued to increase for the next 5 days. By 36 h, the concentration of polyhedrin increased and was maintained at a high level in the later stages of infection. One-third of this group of infected larvae survived the infection. In these individuals, the concentrations of virus and polyhedrin declined to a low level 5 days after infection. This suggests the presence of a host mechanism for clearing the virus from the hemolymph. Key words: infection mechanism, monoclonal antibody, in vitro immunization, Lymantria dispar nuclear polyhedrosis virus, ELISA. Yu, C.-G., MA, M., DING, T., HETRICK,F., et Hsu, H.-T. 1992. Biochemical characterization and time-course analysis of Lymantria dispar nuclear polyhedrosis virus with monoclonal antibodies. Can. J . Microbiol. 38 : 248-257. Des lignees cellulaires d'hybridomes, qui secretent des anticorps monoclonaux (MAbs), ont ete developpees pour reconnaitre specifiquement une proteine virale de 31 000 ou une proteine de polyedrine de 31 000 du virus de la polyedrose nucleaire de Lymantria dispar (LdNPV). Les deux polypeptides se sont montres differents par comparaison de leurs compositions en acides amines. L'immuno-microscopie electronique a ete utilisee pour verifier la liaison specifique des MAbs avec leurs cibles respectives. Les MAbs specifiques ont ete utilises dans une methode de type ELISA qui a ete developpee pour suivre l'evolution du virus LdNPV et de la polyedrine in vivo. Des larves ont ete nourries avec lo6 corps d'inclusions de polyedres. Les resultats ont indique que l'augmentation de la concentfation de virus dans l'hemolymphe a debute 16 h apres l'lnoculation et s'est poursuivie pendant les 5 jours suivants. A 36 h, la concentration de polyedrine a augmente et s'est maintenue a un niveau eleve dans les phases finales de l'infection. Un tiers de ce groupe de larves infectees a survecu a l'infection. Chez ces individus, les concentrations de virus et de polyedrine ont baisse a un niveau faible 5 jours apres l'infection. Ce resultat suggere la presence d'un mecanisme propre a l'h6te pour debarasser le virus de l'hemolymphe. Mots cles : mecanisme d'infection, anticorps monoclonal, immunisation in vitro, virus de la polyedrose nucleaire de Lymantria dispar, ELISA. [Traduit par la redaction]

e en ti on of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable. 2 ~ r e s e naddress: t Department of Zoology, University of Toronto, Ont., Canada M5S 1Al. 3 ~ u t h o to r whom all correspondence should be addressed. Printed in Canada / lmprime au Canada

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Introduction Lymantria dispar nuclear polyhedrosis virus (LdNPV) was first described in 1900 as a causative agent of wilt disease of the gypsy moth (Glaser and Stanley 1943). Most research on the gypsy moth nuclear polyhedrosis virus (NPV) in recent years has been directed toward the use of this virus as a biological insecticide for the control of gypsy moth (Altenkirch et al. 1986; Weseloh 1986; Skatulla 1986; Beck 1985; Obenchain 1985; Abrahamson and Eggen 1985). Although LdNPV has been registered in 1978 as a biological pesticide for the control of gypsy moth (Gypchek) in the United States and Canada (Bell et al. 1981), little is known about the infection mechanisms and pathophysiology of this virus. In previous biochemical studies of LdNPV, there is a lack of consensus regarding the protein composition of this virus. Maskos and Miltenburger (1981) reported the identification of 20 structural proteins of LdNPV by SDS-PAGE when stained with Coomassie Blue. Stiles et al. (1983) reported that 29 structural proteins of LdNPV were identified using SDS-PAGE and silver staining. In addition to the differences in gel electrophoresis protocols, McCarthy and Lin (1976) have shown that various conditions of alkaline dissociation of polyhedra also resulted in different banding patterns using SDS-PAGE. Most immunological studies of LdNPV focused on its relationships with other similar viruses. A few papers examined the serological relationship between the different protein components of LdNPV (McCarthy and Lambiase 1979; Peter and Dicapua 1978; Norton and Dicapua 1975). Norton and Dicapua (1975) indicated that the polyhedrin of LdNPV was immunologically cross-reactive with the viral proteins in a hemagglutination test. Peter and Dicapua (1978) demonstrated serological cross-reactivities between polyhedrin and virus particles of LdNPV prepared by density-gradient centrifugation, using a hapten inhibition test. McCarthy and Lin (1976) reported two or ,three faint precipitin bands when an excess amount of LdNPV virus was assayed against a polyhedrin antiserum in an immunodiffusion test. It was not possible to conclude from these studies whether the cross-reactivities were due to impurities or the presence of similar epitopes in polyhedral and viral proteins. Only a few reports dealt with the infective process of LdNPV using biochemical and ultrastructural methods (McClintock et al. 1986; Caloianu et al. 1978; Bakhvalov et al. 1982; Shield 1984). Monoclonal antibodies (MAbs) and polyclonal antibodies have been used successfully in immunochemical assays for monitoring virus infection. MAb has the additional advantage of having its affinity directed toward a single antigenic determinant, thus avoiding cross-reactions that could occur with polyclonal antibody. In this study, we used both in viva and in vitro immunization procedures for the production of MA^^ against both and polyhedra' proteins of LdNPV and these MAbs in an studying the 'Ourse of LdNPV infee tion in the gypsy moth larval host. Materials and methods Insect rearing Gypsy moths (Lymantria dispar strain NJSS) were received as fourth-instar larvae from Dr. Robert Bell of the Insect Reproduc-

a

Wavelength (220320 nm)

FIG. 1. (a) Rate zonal sedimentation profile of LdNPV virus preparation in 20-60% (w/w) sucrose gradients. (b) UV adsorption spectrum of virus preparation from 20-60% sucrose gradient purification. Numbers represent samples from gradient fractions 1, 2, 3, 4, and 5 indicated in Fig. la. tion Laboratory, USDA, Agricultural Research Service, Beltsville, Md., U.S.A. The larvae were maintained on a modified wheatgerm diet (Bell et a/. 1981) at 26°C under a 16 h light : 8 h dark photoperiod. Polyhedral inclusion body (PIB) purification LdNPV (Hamden isolate) PIBs were washed three times with water, layered onto a linear 40-63% (w/w) sucrose gradient, and centrifuged at 7000 x g at 5°C for 1 h. The PIB layers in the gradient were identified by dispersed light. Each layer was collected, diluted with water, and pelleted by centrifugation at 7000 x g for an additional 10 min. The resultant pellet constituted purified PIBs (McCarthy and Lin 1976). Purification of virus particles Virus particles were released from polyhedra by using the methods described by Bergold (1947), with minor modifications. The purified PIBs were dissociated in a pH 10.8 buffer (0.1 M Na,C03 and 0.17 M Nacl) at room temperature for 15 min. Reaction was terminated by lowering the pH to 8.5 with a pH 8 buffer (0.05 M Tris). Undissolved polyhedra and precipitating polyhedrin were removed by centrifugation at 7000 x g for 15 min. The supernatant was removed and centrifuged again at 42 000 x g for 1 h. The pellet was resuspended in distilled water, layered on the top of a 20% (w/w) sucres; solution, and centrifuged at 100 000 x for 1 h. The pellet was resuspended again in distilled water, layered on a linear sucrose gradient solution (40-60% w/w), and centrifuged

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CAN. J . MICROBIOL. VOL. 38, 1992

FIG. 2. Electron micrographs of LdNPV purified by sucrose gradient centrifugation. Fractions were dialyzed against distilled water, concentrated by speed vacuum concentration, and negatively stained by 1Yo phosphotungstic acid. (a) No virus particles were evident in fraction 1. (b) Fraction 2 contained enveloped single nucleocapsids. (c) Fraction 3 contained enveloped nucleocapsid doublets. (d) Fraction 4 contained enveloped nucleocapsid triplets. (e) Fraction 5 contained unenveloped or naked nucleocapsids. Bars represent 300 nm. at 100 000 x g for 1 h. The virus fractions were collected by an ISCO fractionator, model 640 (Instrumentation Specialist Co., Lincoln, Nebr., U.S.A.). Each fraction was diluted with 20 mL of water and centrifuged at 42 000 x g to remove sucrose.

Tris-HC1 at pH 6.8. Samples were electrophoresed in a buffer containing 0.025 M Tris, 0.192 M glycine, and 0.1 % SDS, at pH 8.3. Gels were stained with Coomassie Blue for 1 h, and destained in 7% acetic acid and 30% methanol.

Polyhedrin purification Polyhedrin was purified according to a procedure described by Summers and Smith (1978). Purified PIBs in water were incubated at 70°C for 2 h before centrifugation at 7000 x g for 10 min. Pellets were then resuspended in a pH 7.8 buffer (0.01 Tris, 0.01 M HgCl,) overnight at room temperature to inactivate proteases. PIBs were dissolved by adding a pH 10.8 buffer (0.1 M Na2C03, 0.17 M NaCl) for 10 min at 4"C, and the solution was centrifuged at 10 000 x g for 1 h at 4°C. The supernatant contained purified polyhedrin.

Amino acid analysis of 31 000 molecular weight virus polypeptide and 31 000 molecular weight polyhedrin The SDS-PAGE gel strips, corresponding to 31 000 molecular weight (MW) polyhedrin and 31 000 MW virus polypeptide bands, were excised and liquified by pushing them through 5-mL syringes. The gels were allowed to sit in a pH 8.9 buffer (0.1 M Tris-HC1) for 24 h at 4°C and filtered through No. 1 Whatman paper. This procedure was repeated three times. Filtrates were pooled and dialyzed against distilled water. The 31 000 MW polyhedrin and 31 000 MW viral polypeptide samples were then concentrated by a Savant speed vacuum centrifuge (model RT-100A, Savant Instruments Inc., Hicksville, N.Y., U.S.A.). Amino acid composition analysis of the two polypeptides was performed by Dr. Tomas Kempe, Protein and Nucleic Acids Laboratory, Department of Chemistry, University of Maryland, College Park, Md., with a model 9153B Aminoquant amino acid analyzer (Hewlett-Packard Co., San Jose, Calif., U.S.A.).

UV absorption spectrum of virus fractions The virus and polyhedrin fractions from the above purifications were dialyzed against distilled water to remove residual sucrose. Preparations were then scanned by UV light from 220 to 320 nm with a UV-visible recording spectrophotometer (model UV-286 Shimadzu Co., Kyoto, Japan). SDS-PA GE gel electrophoresis Virus preparations were analyzed using SDS-PAGE with a minivertical 11% polyacrylamide gel (Ideal Scientific Inc., Rockville, Md., U.S.A.). The running gel buffer contained 0.15 M Tris-HC1 at pH 8.8, and the stacking gel buffer contained 0.05 M

Development of polyclonal antibodies to virus Guinea pigs were subcutaneously immunized with the purified virus and polyhedrin preparations. The primary injection contained 200 pg of the purified antigen emulsified in complete Freund's

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YU ET AL.

FIG. 3. SDS-PAGE of virus of polyhedrin of LdNPV. Lanes 1 and 9, molecular weight markers; lane 2, polyhedrin; lane 3, virus after 20% sucrose purification; lane 4, crude virus preparation; lanes 5-8, virus from fraction 2, 3,4, and 5, respectively, after 20-60% sucrose gradient purification. adjuvant. Two weeks later, 200 pg antigen in incomplete Freund's adjuvant was given, and in another week, a second booster of 200 pg antigen in phosphate-buffered saline (PBS) was administered. Ten days after the final injection, blood was collected by cardiac puncture and the serum collected and stored at -20°C for future use. Immunoglobulin G was purified from guinea pig antiserum, using a protein A CL-4B column (Pharmacia Fine Chemicals Co., Sweden) (Ey et al. 1978). Development of MAbs to polyhedrin and virus Twenty percent sucrose gradient purified virus and purified polyhedrin were used as immunogens in different cell fusions. For in vivo immunization, 6-week-old BALB/c mice were given three intraperitoneal injections, 50 pghnjection, according to procedures described previously for the immunization of guinea pigs. In vitro immunization was conducted as described by Ma et al. (1984). Five micrograms of 20-60% sucrose gradient purified virus particles was used as immunogen. Ten days after the primary injection, spleen cells were released into RPMI 1640 medium + 10% fetal calf serum + 10% EL-4 thymoma cell conditioned medium. One microgram of purified virus and added into this cell culture medium for in vitro immunization of the B-cells. After 4 days, the spleen cells were harvested for cell fusion. Cell fusion, hybridoma colony propagation, and screening procedures after in vivo and in vitro immunizations were according to procedures described by Wu and Ma (1985). Spleen cells were mixed with FOX-NY myeloma cells as fusion partners at the ratio of 5: 1, and 50% PEG 1450 (Kodak) was used as fusion medium. Enzyme-linked immunosorbent assay (ELISA) and Western blot The same procedure was used for monitoring of viruses and polyhedrin. (i) The ELISA plate was first coated with 100 pL per well (10 pg/mL) guinea pig anti-virion or anti-polyhedrin immunoglobulins overnight at 4°C. (ii) The wells were then blocked with 2% bovine serum albumen (BSA) in pH 7.3 PBS for 1 h before adding the antigens (virus, polyhedrin, and hemolymph). (iii) After washing the plate three times with PBS, MAb (10 pg/mL) was added for 1 h. (iv) MAb solutions were decanted, washed three times with PBS, followed by the addition of horseradish peroxidase labeled goat anti-mouse IgG + IgM. (v) After the plate was exten-

97 000 66 000

@

42 000

30 000

FIG. 4. SDS-PAGE purification of 31 000 MW polyhedrin and 31 000 MW viral polypeptide. Lanes 1 and 4, molecular weight markers; lane 2, 31 000 MW polyhedrin protein; lane 3, 31 000 MW viral protein. sively washed with PBS, 100 pL of the substrate 2,2'-azinodi(3-ethylbenzthiazoline sulfonate) (ABTS) + hydrogen peroxide (Kirkegaard & Perry Laboratory, Gaithersburg, Md., U.S.A.) was added. The optical density readings were recorded at 405 nm with a microplate reader (model MR 600, Dynatech Instrument Inc., Torrance, Calif., U.S.A.). To verify the binding of MAbs to viral proteins were transferred from slab SDS-PAGE gels to nitrocellulose membranes (Bio-Rad Laboratories, Richmond, Calif., U.S.A.), followed by incubation with MAb and visualization by enzyme-labeled secondary antibody, according to the procedure of Towbin et al. (1979). Monitoring the development of virus andpolyhedrin in hemolyrnph Thirty larvae were given lo6 PIBs/larva per 0s. Aliquots (100 pL) of PIBs were delivered to the foregut region of the larvae by a smoothend 18-gauge needle. At different time intervals, larvae were placed in a - 20°C freezer for 30 s until they were sluggish and easy to handle. The larvae were pierced at the end of a proleg

CAN. J . MICROBIOL. VOL. 38, 1992

TABLE1. Amino acid analysis (number of amino acid residues) of polyhedrin and viral proteins that possess similar molecular weight

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Amino acid residues

1

VIRUS

I

Vlrus and polyhedrln concentratlon (pg/mL)

Polyhedrin protein (3 1 000 MW)

Virus protein (3 1 000 MW)

Asx Glx Ser His G~Y Thr Ala TY~ Cys-Cys Val Met Leu P he LYs Pro Total MW NOTE: SDS-PAGE purified 31 000 MW virus polypeptide and 31 000 MW polyhedrin were hydrolyzed in 6 M HCl, converted to fluorescent OPA derivatives, separated with HPLC, and analyzed on a Hewlett-Packard Aminoquant amino acid analyzer.

with a 22-gauge needle. A 5-pL hemolymph sample was collected with a capillary pipet, transferred into 100 pL PBS saturated with phenylthiourea, and stored at - 70°C. When the bleeding had stopped after hemolymph collection, each larva was returned to a 25°C incubator for later bleedings. Hemolymph samples from five larvae that died from infection on day 8 or 9 and five larvae that survived were tested by ELISA for virus and polyhedrin concentrations.

Vlrus and polyhedrln concentratlon (pg/mL)

VIRUS AND HEMOLYMPH

-

HEMOLYMPH

concentratlon of vlrus (pg/mL) F I G . 5. (a) A dose-response curve demonstrating the affinity of F3V-1C4 to purified polyhedrin and virus in an indirect ELISA test. (b) A dose-response curve demonstrating the affinity of F1V-2C11 to purified virus and polyhedrin in an indirect ELISA test. (c) Reactivity of F1V-2Cl1 MAbs with normal gypsy moth hemolymph (1: 10 in PBS) mixed with a purified virus preparation. FlV-2C11 antibodies did not react with gypsy moth hemolymph.

Electron microscopy Gypsy moth fat bodies were fixed in 2.5% glutaraldehyde in a pH 7.4 0.12 M sodium phosphate buffer for 24 h at 4°C. They were washed three times and postfixed in 1% osmium tetroxide for 1.5 h. The tissues were then dehydrated in a series of ethanol solutions and embedded in LR-White resin (Polysience Inc., Warringnton, PA., U.S.A.). Resin blocks were sectioned with a LKB Ultratone I11 (LKB Biotechnology Inc., Piscatway, N.J., U.S.A.). Grids with thin sections were incubated in a blocking solution (2% BSA, 1% goat serum in PBS) for 1 h, followed by incubation in the primary antibody (1 :10 supernatant of cell line in 1% goat serum in PBS) at room temperature for another 2 h. After washing three times with PBS, sections were incubated with the secondary antibody (gold-labeled goat anti-mouse IgG + IgM) at room temperature for 1 h. Sections were washed three times with PBS, two times with distilled water, and then stained with 1% uranyl acetate followed by 1% lead citrate. The stained sections were observed in a Zeiss 10-CA transmission electron microscope (Carl Zeiss Co., Germany).

Results Purification of alkaline-dissociated L d N P V Sucrose density gradient (20-60%) centrifugation of virus preparations resulted in a multiple-banded sedimentation profile (Fig. la). It revealed five distinct absorbance peaks. ratio was obtained for material collected The A260/A280 from each peak (Fig. lb). The ratio of peak 1 was 1.00, whereas the values for peaks 2 , 3 , 4 , and 5 ranged from 1.27 to 1.30. Peak 1 near the top of the gradient did not contain virus particles when examined by electron microscopy (Fig. 2a). Peaks 2, 3, and 4 contained single, double, and

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YU ET AL.

FIG. 6 . Immuno-electron microscopy of LdNPV-infected fat body. The dark dots indicated by arrows are immunogold particles. (a) Ultrathin section treated with F1V-2C11 as secondary antibody. A total of 96.5% of gold particles on polyhedra bound to polyhedrin matrix. ( b ) Ultrathin section treated with F1V-1C4 as secondary antibody. Gold particles bound exclusively to virus particles. Bar represents 300 nm for Figs. 6a and 6b.

triple enveloped nucleocapsids, respectively (Figs. 2b, 2c, and 2d). Peak 5 contained unenveloped nucleocapsids (Fig. 2e). SDS-PAGE analysis of polyhedrin and virus Virus particles from the 20-60% sucrose gradient centrifugation, the 20% sucrose sedimentation, crude preparation, and polyhedrin were subjected to SDS-PAGE analysis (Fig. 3). The polyhedrin sample consisted of a predominant band with a molecular weight of 3 1 000 (Fig. 3,

lane 2). The SDS-PAGE patterns of virus from peaks 2, 3, 4, and 5 were identical (Fig, 3, lanes 5-8) and were similar to that of the virus preparation from the 20% sucrose centrifugation pellets. They were, however, significantly different from that of the crude preparation (Fig. 3, lane 4). The 20% sucrose centrifugation step apparently removed a substantial portion of the 3 1 000 MW polyhedrin (Fig. 3, lane 3), while the 20-60% sucrose gradient purification step removed most of the soluble proteins from .the virus particles (Fig. la).

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CAN. J. MICROBIOL. VOL. 38, 1992

Days after lnoculatlon

FIG. 8. Time course of development of LdNPV virus and polyhedrin in gypsy moth larvae that were fed lo6 PIBs and died on day 8 or 9. Hemolymph of five infected gypsy moth larvae was collected at different time intervals, and an ELISA with FlV-2C11 and F3V-1C4 was used to determine the concentrations of virus and polyhedrin respectively. Bars represent standard error.

FIG. 7. Western blot analysis of MAbs produced by hybridomas F1V-2C11 and F3V-1C4. Crude virus preparation (15 pg) was applied to each sample well and electrophoresed in 11% SDSPAGE, transferred to nitrocellulose, and reacted with 1:3 dilutions of fluid from cell cultures F3V-1C4 (lane 1) and F1V-2C11 (lane 2). Binding of mouse antibodies was revealed by reaction with alkaline phosphatase labeled goat anti-mouse IgG + IgM and the substrate BCIP-NBT.

h mi no acid composition of 31 000 M W viral polypeptide

and 31 000 M W polyhedrin After the SDS-PAGE purified 31 OoO MW polyhedrin and 31 000 MW viral polypeptide were subjected to SDSPAGE for a second time ( ~ i 4), ~ .amino acid analysis revealed two distinct amino acid compositions (Table 1). The 31 000 MW viral protein consisted of 33 alanine, 60 proline, 12 serine, 29 isoleucine, and no methionine residues, whereas the 31 000 MW polyhedrin contained 20 alanine, 14 proline, 6 serine, 15 isoleucine, and 6 methionine residues. -

Hemolymph collected from fifth-instar gypsy moth, diluted 1:10 in PBS, was mixed with purified virus; the slope of the dose-response curve of F1 V-2C 11 and the virus was not significantly altered (Fig. 5c). Immuno-electron microscopy (IEM) studies further confirmed the specific binding of F l V-2C 11 MAbs to virus particles. Only virus particles in thin sections of fat body of infected gypsy moths were tagged with gold particles, demonstrating the linkage of immunoglobulins to the target sites (Fig. 6b). A similar study with F3V-1C4 showed that of the 85 gold particles that tagged the polyhedra, 96.5% appeared to be associated with the polyhedrin matrix (Fig. 6a). The three particles that appeared to be linked to virus particles could be incidental association. Western blot analysis showed that F1V-2C11 MAbs reacted prominently with a 31 000 MW viral polypeptide, with minor bands at 21 000 and 15 000 (Fig. 7). F3V-1C4 MAbs were specific only to the 31 000 polyhedrin protein.

. -

Production and characterization of MAbs to viral and polyhedral proteins Most of the positive clones produced MAbs that bound to both polyhedral and viral proteins. Only after extensive screening were two hybridoma cell lines selected on the basis of antibody production to polyhedrin and virus, respectively. F3V- 1C4 derived from in vivo immunization produced MAbs to 31 000 MW polyhedrin, whereas F1V-2C11, obtained from an in vitro immunization, produced MAbs to the 3 1 000 MW viral polypeptide. F3V-1C4 did not react with viral proteins (Fig. 5a), and F1V-2C11 did not bind to polyhedrin (Fig. 5b). Immunoglobulin subclasses of F3V-1C4 and F1 V-2C 11 were determined to be IgG, and IgG2,, respectively .

Temporal development of virus and polyhedrin in hemolymph Thirty fourth-instar gypsy moth larvae were inoculated with lo6 PIBs/larva. By 9 or 10 days after inoculation, 20 died of infection, while 10 survived and developed into pupae. Daily hemolymph samples were collected from all larvae that eventually died from infection after ingesting lo6 PIBs/larva. Five larvae that died on day 8 or 9 were selected for ELISA analysis of polyhedrin and virus concentrations. Small peaks in both the virus and polyhedrin development curves were seen 2 h after inoculation (Fig. 8) and probably represented the inocula virus. Virus concentration in the hemolymph began to increase 16 h after inoculation and continued to increase up to day 5. Thirty-six hours after inoculation, polyhedrin concentration in the hemolymph had increased markedly and was maintained at a high level in the later stages of infection. Virus and polyhedrin development in larvae which recovered from infection The concentrations of virus and polyhedrin in the hemolymph of survivors were monitored by ELISA with

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Days after lnoculatlon

Days after lnoculatlon FIG. 9. Time course of development of LdNPV virus and polyhedrin in gypsy moth larvae that were fed lo6 PIBs and survived the infection. Hemolymph samples of five such individuals were collected at different time intervals. (a) Virus development was monitored by ELISA with F1V-2C11. Continuous line indicates virus development in the larvae that died of infection. Broken line indicates virus development in the hosts that recovered from infection. (b) Polyhedrin concentrations in the hemolymph were monitored by ELISA with F3V-1C4. Continuous line indicates polyhedrin development in the hosts that died of infection. Broken lines indicates polyhedrin development in the hosts that recovered from the infection. Bars represent standard error.

MAbs F1V-2C 11 and F3V- 1C4, respectively (Figs. 9a and 9b). The time course of virus development in survivors was different from those that died from infection (Fig. 9a). One day postinoculation, virus concentration in these larvae began to rise and continued up to day 5. Unlike in those larvae that died from infection, virus concentration in survivors began to decrease starting 30 h after inoculation, and by day 5 virus concentration had declined to an extremely low level. A similar phenomenon was observed when polyhedrin titer was monitored by ELISA (Fig. 9b). Two days after inoculation, polyhedrin concentration began to decrease, and a steady decline was observed during later stages of infection. All survivors completed their life cycle in an apparently normal manner.

255

Discussion SDS-PAGE protein profiles have been useful for identification of different NPVs (Stiles et al. 1983; Maskos and Miltenburger 1981; McCarthy and Lin 1976; Summer and Smith 1978). However, different PAGE protein profiles of LdNPV have been reported. Discrepancies could be attributed to variations in gel electrophoresis protocols and possibly inadequate purification procedures. Additional parameters are clearly necessary to characterize LdNPV. In this study, purification steps were carefully monitored by to ensure pure both electron microscopy and A260/A280 polyhedrin and virus preparations. In addition, two hybridoma cell lines, one each producing MAbs specific to the 31 000 MW viral polypeptide and the major 31 000 MW polyhedrin, respectively, were generated. The ELISA developed using these MAbs was sensitive enough for monitoring polyhedrin and virus development in individual gypsy moth larvae. Amino acid analysis of specific proteins in a NPV could be further explored as a legitimate criterion for species identification. In this study, the amino acid compositions of 3 1 000 MW polyhedral and viral polypeptides were obtained from purified preparations. If amino acid composition could be obtained from polyhedrin of other lepidopteran NPVs, feasibility of this parameter as a species character could then be evaluated. Among the serological methods used in baculovirus studies, ELISA is one of the most rapid and sensitive procedures for the detection of baculovirus antigens (McCarthy and Henchal 1983; Kelly et al. 1976; Longworth and Carey 1980; Crook and Payne 1980). Our objective was to develop an ELISA for the specific detection of polyhedrin and virus. F1V-2C11 and F3V-1C4 were shown to be target specific to virus and polyhedrin, which would permit the detection of different stages of LdNPV development, such as the occluded versus nonoccluded morphotypes (Figs. 5a and 5b). Since normal gypsy moth hemolymph did not interfere with the dose-response curve of F 1C-2C 11 (Fig. 5c), the temporal development of virus in hemolymph of infected larvae can be accurately monitored and quantified. In Western blot analysis, F1V-2C11 reacted to 3 1 000, 25 000, and 17 000 MW virion polypeptides, but not with 3 1 000 MW polyhedrin. This is additional evidence that supports the distinct identities of the 31 000 MW polyhedral and 31 000 MW virion polypeptides. The smaller 25 000 and 17 000 MW viral polypeptides could be the degradative products of the 31 000 MW viral polypeptide. Specificity of the two MAbs developed in the current studies enables the study of temporal and spatial development of the virus and polyhedrin in gypsy moth larvae by ELISA. Protein synthesis in a gypsy moth cell line infected with LdNPV was detected by pulse-labeling experiments (McClintock et al. 1986). However, this method could not distinguish viral proteins from host proteins formed in response to the infection. Present studies reveal that virus concentration in hemolymph began to increase 16 h after inoculation, and polyhedrin concentration began to increase 36 h after inoculation (Fig. 8). The developmental time curves of virus and polyhedrin in gypsy moth larvae were compared with the growth curve of LdNPV in gypsy moth cell line IPLB-LD-652-Y (McClintock et al. 1986). In gypsy moth cell lines, polyhedral inclusion bodies (PIBs) appeared 48 h

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after inoculation. This study shows that polyhedrin began t o be synthesized in infected larvae 36 h after inoculation, approximately 10 h before formation of PIBs in the gypsy moth cell line. Two hours after inoculation, there was a small peak in both virus and polyhedrin concentrations (Fig. 8). They probably represented the appearances of the inoculum virus in the hemolymph after inoculation. It is commonly accepted that replication of NPV first occurs in the midgut epithelial cells before virus enter into the hemocoel. However, Granados and Lawler (1981) showed that direct passage of inoculum virus through midgut epithelial cell cytoplasm and into the hemocoel could occur with Autographa californica NPV (AcNPV) in T. ni larvae. Our results suggest that this pathway is also possible in gypsy moth larvae. The time of appearance of virus and polyhedrin of LdNPV after inoculation is longer than that of AcNPV. In AcNPV-infected larvae, virus appeared 8 h after inoculation and PIBs appeared 16 h after inoculation (Granados and Lawler 1981). It was not determined whether prolongation of the replicative cycle of LdNPV in larvae host was specified by the virus, the host, or by an interaction between the two. There have been several reports on the effects of NPV on survival of host insects. Pupal weight, adult emergence, mean longevity, mean matings per female, or mean eggs laid did not change in surviving T. ni larvae (Igonoffo 1964; Vail and Hall 1969). Bakhvalov et al. (1982) reported that some gypsy moth larvae infected with NPV could complete their development and become adults. Our experiments have shown that virus and polyhedrin development in those larval survivors from LdNPV infection were different from those that died from infection (Figs. 9a and 9b). One day after inoculation, virus concentration in the larvae from those dying of infection continued t o increase. However, in the larvae that survived infection, the virus titer began t o decrease starting from 30 h, and by 5 days after inoculation, virus concentration in the larvae declined t o a low level (Fig. 9a). A similar phenomenon was observed in the time course of polyhedrin (Fig. 9b). One possible mechanism is at the midgut tissue level (Tinsley 1975, 1979). However, we have no evidence t o support this hypothesis at present. Another possibility is the production of an anti-viral humoral factor in response to infection. Aizawa (1953, 1954a, 1954b) and Yamafuji et al. (1958) reported the presence of a proteinaceous inhibitory substance in the hemolymph of NPV-infected silkworm (Bombyx mori) larvae. Tinsley (1975) suggested that if defense mechanisms exist in the hemocoel, they d o not operate immediately, since the virus dosage necessary to cause death by injection into the hemocoel is minimal compared with dosages required for death by ingestion. Another possibility is that the susceptibility of gypsy moth larvae varies among individuals. Development of this sensitive MAb-based ELISA for monitoring viral development will be useful for future studies of insects' defensive mechanisms to NPVs. In conclusion, two hybridoma cell lines specific to 31 000 M W viral protein and 31 000 M W polyhedrin, respectively, were developed and utilized in an ELISA for monitoring viral development in host hemolymph. Amino acid composition analysis revealed that the 31 000 M W viral protein and 31 000 M W polyhedrin were two different polypeptides. Temporal differences in development of virus

and polyhedrin in hemolymph were established in infected larvae. Gypsy moths that survived the infection were shown t o clear the virus from their hemolymph over a period of 3-5 days. Acknowledgements This research was supported by the College of Agriculture and Life Sciences, University of Maryland at College Park, USDA Cooperative Agreement 58-32U4-2-375, USDA Competitive Grant 85-FSTY-9-0118, and Maryland Agricultural Experiment Station scientific artcle A61 10, contribution No. 8275. Abrahamson, L.P., and Eggen, D.A. 1985. Recent field research using microbial insecticide against gypsy moth. USDA For. Serv. Gen. Tech. Rep. NE, 1985(100). pp. 95-98. Altenkirch, W., Huber, J., and Krieg, A. 1986. Field trial for microbial control of Lymantria monacha L. J. Plant Dis. Prot. 93: 479-493. Aizawa, K. 1953. Sedimentation of the silkworm jaundice virus by the ultracentrifuge. I. Effect of the vaccine made from sediment. Jpn. J. Appl. Zool. 18: 141-142. Aizawa, K. 1954a. Immunological studies of the silkworm jaundice virus. 11. Agglutination reaction of the polyhedral bodies. Virus (Osaka), 4: 241-244. Aizawa, K. 1954b. Immunological studies of the silkworm jaundice virus. 111. Experiments on the defense of infection in the silkworm jaundice. Virus (Osaka), 4: 245-248. Bakhvalov, S.A., Larionov, G.V., and Bakhvalova, V.N. 1982. Recovery of Lymantria dispar Lepidoptera Lymantriidae after experimental virus infection. Entomol. Rev. 61(4): 755-758. (Engl. Transl. from Entomol. Obozr.) Beck, W.R. 1985. Recent development in the Zoecon Corporation and the Thuricide forestry formulation. USDA For. Serv. Gen. Tech. Rep. NE, 1985(100). pp. 14.1-142. Bell, R.A., Owen, C.D., Shapiro, M., and Tardif, J.R. 1981. Development of mass-rearing technology. In The gypsy moth: research towards integrated pest management. Edited b y C.C. Doane and M.L. McManus. U.S. Department of Agriculture, Washington, DC. 1548. pp. 599-693. Bergold, G. 1947. Die Isolierung des Polyeder-Virus and die Natur der Polyeder. Z. Naturforsch. 2b: 122-143. Caloianu, M., Saftoiu, D., and Mihalache, G. 1978. Electron microscopic studies on the larvae of Lymantria dispar naturally infected by the virus of nuclear polyhedrosis BorrelinavirusReprimens. Zast. Bilja, 29: 57-67. Crook, N.E., and Payne, C. 1980. Con~parisonof three methods of ELISA for baculoviruses. J. Gen. Virol. 46: 29-37. Ey, P.L., Prowse, S. J., and Jenkin, C.R. 1978. Isolation of pure IgG,, IgG,,, and IgG,, immunoglobulins from mouse serum using protein A - Sepharose. Immunochemistry, 15: 429-436. Glaser, R. W., and Stanley, W.M. 1943. Biochemical studies on the virus and inclusion bodies of silkworm jaundice. J. Exp. Med. 77: 45 1-466. Granados, R.R., and Lawler, K.A. 1981. In vitro pathway of Autographa californica baculovirus invasion and infection. Virotology, 108: 297-308. Ignoffo, C.M. 1964. Bioassay techniques and pathogenicity of a nuclear polyhedrosis virus of the cabbage looper, Trichoplusia ni (Hubner). J. Insert Pathol. 6: 237-245. Kelly, D.C., Edward, M.L., Evans, H.F., and Robertson, J.S. 1976. The use of enzyme-linked immunosorbent assay to detect a nuclear polyhedrosis virus in Heliothis amigera larvae. J. Gen. Virol. 40: 456-469. Longworth, J.F., and Carey, G.P. 1980. The use of an indirect enzyme linked immunosorbent assay to detect baculovirus in larvae and adults of Oryctes rhinocerus from Tonga. J. Gen. Virol. 47: 431-438.

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Ma, M., Wu, S.J., Howard, M., and Borkovec, A.B. 1984. Enhanced production of mouse hybridoma to picomoles of antigen using EL-4 conditional media with an in vitro immunization protocol. In Vitro, 20: 730-742. Maskos, C.B., and Miltenburger, H.G. 1981. SDS-PAGE comparative studies on the polyhedral and viral polypeptides of the nuclear polyhedrosis viruses of Mamestria brassicae, Autographa culifornica and Lymantria dispar. J . Invertebr. Pathol. 37: 174- 180. McCarthy, W.J., and Henchal, L.S. 1983. Detection of Autographa californica baculous nonoccluded virion in vitro and in vivo by enzyme-linked immunosorbent assay. J. Invertebr. Pathol. 4,l: 401-404. McCarthy, W. J., and Lambiase, J.T. 1979. Serological relationships among Pulsiinae baculoviruses. J. Invertebr. Pathol. 34: 170-177. McCarthy, W.J., and Lin, S. 1976. Electrophoretic and serological characterization of Porthetria dispar polyhedrin protein. J. Invertebr. Pathol. 28: 57-65. McClintock, J.T., Dougherty, E.M., and Weiner, R.M. 1986. Protein synthesis in gypsy moth cell infected with a nuclear polyhedrosis virus of Lymantria diapar. Virus Res. 5: 307-322. Norton, P . W., and Dicapua, R.A. 1975. Serological relationship of nuclear polyhedrosis virus. J. Invertebr. Pathol. 25: 185-1 88. Obenchain, F.P. 1985. Commercial production of microbials by Reuter Laboratories, Inc., for control of the gypsy moth and the spruce budworm. USDA For. Serv. Gen. Tech. Rep. NE, 1985(100). pp. 139-140. Peter, J.E., and Dicapua, R.A. 1978. Immunochemical characterization of Lymantria dispar polyhedrosis virus hemagglutinin protein carbohydrate interaction. Intervirology, 9: 231-242.

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Shield, K.S. 1984. Proceeding of the 42nd Annual Meeting of the Electron Microscopy Society of America. pp. 222-223. Skattula, U. 1986. On the susceptibility of Lymantria dispar (Lepidoptera, Lymantriidae) larvae of different geographical regions to a nuclear polyhedrosis virus. J . Appl. Entomol. 103: 203-208. Stiles, B., Burand, J.P., Meda, M., and Wood, H.A. 1983. Characterization of gypsy moth (Lymantria dispar) nuclear polyhderosis virus. Appl. Environ. Microbiol. 46: 297-303. Summer, M.D., and Smith, G.E. 1978. Baculovirus structural polypeptides. Virology, 84: 390-402. Tinsley, T.W. 1975. Factors affecting virus infection of insect gut tissue. In Invertebrate immunity. Edited by K. Mararnorosch and R.E. Shope. Academic Press, New York. pp. 55-63. Tinsley, T.M. 1979. The potential of insect pathogenic viruses as pesticidal agents. Annu. Rev. Entomol. 24: 63-88. Towbin, H.S., Staehelin, T., and Gordon, J. 1979. Electrophoresis transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sc. U.S.A. 76: 4350-4354. Vail, P.V., and Hall, I.M. 1969. Susceptibility of the pupa of the cabbage looper, Trichoplusia ni to nuclear polyhedrosis virus. I. General response. J . Invertebr. Pathol. 14: 227-236. Weseloh, R.M. 1986. Laboratory assessments of forest microhabitat substrate as source of gypsy moth nuclear polyhedrosis virus. J . Invertebr. Pathol. 48: 27-33. Wu, S.J., and Ma, M. 1985. In vitro immunization with EL-4 conditioned medium for hybridoma production. J. Tissue Cult. Methods, 3: 147-149. Yamafuji, K., Omura, H., and Otoma, N. 1958. An immunological investigation of viral polyhedrosis. Enzymologia, 19: 175-179.