Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

JOURNAL

OF [NVERTEBRATE

Site of Action

PATHOLOGY

of a Synergistic Factor of a Granulosis Armyworm, Pseudaletia unipuncta

Y. TANADA, Division

34, 249-255 (1980)

of Entomology

H. INOUE,’ and

T. HESS, AND ESTHER M. OMI

ROBERTA

Parasitology,

Virus of the

University

of California,

Berkeley,

California

94720

Received May 23, 1979 A synergistic factor (SF), which is present in the capsule matrix protein of a granulosis virus of the armyworm, Pseudaletia unipuncta, enhances haculovirus infection in armyworm larvae. The site of action of the SF was investigated. The oral inoculation of SF did not enhance the infectious hemolymph virions which had been inoculated into the hemocoel. The SF also did not enhance the infection of purified enveloped virions when both virus and SF were inoculated into the hemocoel, but enhancement occurred when they were inoculated orally. Thus, the activity of the SF was confined to the midgut lumen. Observations with ferritin-conjugated antibody indicated that the site of action of SF was the cell membrane of the microvillus. There were more ferritin particles attached to midgut cell membranes of larvae inoculated orally with SF than to those of control larvae inoculated with buffer. KEY WORDS: armyworm; Pseudaletia unipuncta; synergistic factor, site of action; baculovirus; granulosis virus; nuclear polyhedrosis virus: ferritin-conjugated antibody; larval midgut; microvillus, cell membrane of. -

INTRODUCTION

the use of purified SF rather than the GVH capsule and by the ferritin-antibody technique .

The Hawaiian strain of a granulosis virus (GVH) interacts synergistically with a nuclear polyhedrosis virus (NPV) in the larva of the armyworm, Pseudaletia unipuncta (see Tanada, 1956, 1959). The GVH contains an enhancing factor in the proteinaceous matrix of the capsule (occlusion body) (Tanada and Hukuhara, 197 1; Tanada et al., 1973; Hara et al., 1976). The site of action of the synergistic factor (SF) appeared to be the midgut because the GVH, when fed to armyworm larvae, did not enhance the NPV which had been inoculated into the hemocoel (Tanada and Hukuhara, 1971). Furthermore, preliminary observations with the electron microscope indicated that the SF enhanced the attachment of enveloped virions to the cell membrane because more vu-ions were attached in larvae fed the SF than in control larvae fed the buffer (Tanada et al., 1975). In the present study, we have further investigated the site of action of the SF with

MATERIALS

AND METHODS

Host insect. The colony of armyworms was started from field-collected adults and had been maintained in the laboratory for several years. Fifth-instar larvae were used in the tests. They were fed an artificial diet (Tanada and Hukuhara, 1971) and maintained individually in plastic jelly wells (Champion Packages Co., Belvidere, Illinois). Two sheets of wells, placed opposite each other, were used to form individual compartments. Larvae under CO, anesthesia were inoculated with 2 ~1 inoculum/larva using a microapplicator (Tanada and Hukuhara, 1971). The oral inoculation was made with a blunted 30-gauge needle. A sharp 30-gauge needle was inserted through the base of an abdominal proleg for intrahemocoelic inoculation. Virus. The baculoviruses were the synergistic Hawaiian strain of a granulosis virus (GVH) and the typical strain of a nuclear

i Present address: Virus Laboratory, Sericultural Experiment Station, 55-30 Wada, 3-chome, Suginamiku, Tokyo 166, Japan. 249

0022-201 l/80/030249-07$01.00/o Copyright All rights

@ 1980 by Academic Press, Inc. of reproduction in any form reserved.

250

TANADA

polyhedrosis virus (NPV), both of the armyworm. Preparation of synergistic factor (SF). The propagation and purification of GVH capsules and the isolation of SF from 200 mg of capsules were according to the method of Hara et al. (1976). Preparation of enveloped virions. The enveloped virions of NPV were liberated from the polyhedra with 0.02 N NaOH and purified by differential centrifugation in alkali according to the method of Yamamoto and Tanada (1978b). Hemocoelic activity of SF. The likelihood of enhancement by orally inoculated SF on the NPV within the hemocoel was investigated in two separate tests. In the first test, two types of NPV inocula were used, the virus-infected hemolymph and the enveloped virions obtained from polyhedra. The NPV-infected hemolymph was obtained from fifth-instar larvae which had been fed 5 days previously with a suspension of 1 x IO5 polyhedra on seedling-corn leaves. The collected hemolymph was kept chilled in an ice-filled bucket until diluted. The hemolymph was diluted 10p3, 10m4, 10e5, and 10v6 with 10 mM Tris-HCl buffer, pH 8. The concentration of SF was 0.5 mg/ml of Tris-HCl buffer. There were 20 larvae per treatment. The treatments are given in Table 1. In the second test, the NPV inoculum was purified enveloped virions obtained from polyhedra. Since a preliminary screening trial indicated that a concentration of 10” enveloped virions/ml resulted in 100% larval infection when orally inoculated, a concentration of lOlo enveloped vu-ions/ml was selected for the oral treatment. For the intrahemocoelic inoculation, less than 100% larval infection occurred at lo7 enveloped virions/ml, and this concentration was selected. There were two replications of the trials. The treatments are given in Table 2. Preparation of immune serum of SF. A mixture of equal volumes of SF and complete Freund’s adjuvant was inoculated

ET

AL.

TABLE EFFECT ORALLY

1

OF SYNERGISTIC FACTOR (SF) INOCULATED ON THE INFECTIVITY OF NPV-INFECTED

HEMOLYMPH INTO THE

WHICH HAD BEEN INOCULATED HEMOCOEL OF FIFTH-INSTAR ARMYWORM

LARVAE”

Treatment Oral inoculation

No. virusinfected larvae

Intrahemocoelic* inoculation

SF SF SF SF Control (water)

NPV, NPV, NPV, NPV, NPV, NPV, -

lo-’ lo-’ 10-j 10-j IO-” lo-”

20 20 10 12 5 4 0 0

RThere were 20 fifth-instar larvae in each treatment. See text for a description of the procedure used in the test. b Preliminary test on the infectivity of NPV in the hemolymph had indicated that dilutions below IO-’ produced 100% mortality.

subcutaneously four times at weekly intervals into a New Zealand white rabbit. The concentration of SF in each inoculum was 0.3 mg of protein as determined by Feulgen phenol reagent. The blood was collected from the rabbit 7 days after the last inocuTABLE EFFECT INFECTIVITY WHICH

OF THE

2

SYNERGISTIC

FACTOR

OF ENVELOPED VIRIONS HAD BEEN INOCULATED AND I~~TRAHEIV~~~OELICALLY”

ON THE OF A ORALLY

NPV

No. virus-infected larvae Treatment”

Trial I

Trial II

Oral, NPV Oral, NPV + SF IH, NPV IH, NPV + SF Oral, control IH, control

8 18 4 4 0 0

11 19 11 12 0 0

’ Each treatment had 20 fifth-instar larvae, except each control group had 10 larvae. * The inocula were applied at 2 pi/larva. The virus concentration for oral inoculation was 10”’ enveloped virions/ml, and that for intrahemocoelic inoculation (IH) was 10’ enveloped virionslml.

ACTION

SITE

OF

VIRUS

lation. The titer of the immune serum against the SF antigen by agar immunodiffusion test was 1:32. Preparation of ferritin solution. The ferritin solution containing 100 mg/ml of 0.15 M NaCl was purchased from Sigma Chemical Co. (St. Louis, Missouri). The ferritin was purified with ammonium sulfate as follows. Five milliliters of the ferritin solution was added to 20 ml of 2% ammonium sulfate and centrifuged at 3000 rpm for 20 min. The collected supernatant was added to 25 ml of saturated ammonium sulfate, pH 7, stirred for 1 hr, and then centrifuged at 3000 r-pm for 20 min. The precipitate was dissolved in 5 ml of distilled water, mixed with saturated ammonium sulfate, and centrifuged as in the previous step. The ferritin precipitate was resuspended in 5 ml of distilled water, dialyzed against 0.05 M phosphate buffer, pH 7.5, for 24 hr, and centrifuged at 35,000 rpm for 2 hr. The precipitate was dissolved in 5 ml of 0.05 M phosphate buffer, pH 7.5. The above procedures were carried out at 5°C. Purification of y-globulin. The y-globulin was purified by salting out with ammonium sulfate according to the method of Kawamura (1969). The y-globulin solution was adjusted to 50 mg/ml of protein volume. Preparation of ferritin-antibody conjugate. A mixture of 150 mg of y-globulin, 2

ml of 4% sodium carbonate, 360 mg offerritin, and 5 mg of p,p’-difluoro-m,m’dinitrodiphenyl sulfone in 1 ml of cold acetone was gently stirred for 24 hr at 4°C. The mixture was then dialyzed against 0.05 M phosphate buffer, pH 7.5, for 24 hr at 5°C and centrifuged at 3000 rpm for 10 min. The supernatant was centrifuged at 40,000 r-pm for 3 hr. The precipitate was suspended in 10 ml of 0.05 M phosphate buffer,pH 7.5, and centrifuged at 40,000 rpm for 3 hr. The last step was repeated two more times with new buffer in order to remove unconjugated y-globulin. The final precipitate was suspended in 10 ml of 0.05 M phosphate buffer, pH 7.5. In order to reduce nonspecific reactions, the conjugated y-globulin was

SYNERGISTIC

251

FACTOR

absorbed with an acetone powder preparation of healthy armyworm larvae as follows. Three milliliters of the suspended conjugate was absorbed onto 300 mg of powdered larval preparation for 1 hr at room temperature (ca. 25°C) and then centrifuged at 10,000 x-pm for 1 hr. The supernatant was diluted to 1: 10 with buffer before use. Localization

of ferritin

in larval

midgut.

The SF, at a concentration of 0.5 mg/ml of 0.05 M Tris-HCl buffer, pH 8, was inoculated orally at a dose of 2 pi/larva. The buffer alone at a dose of 2 $/larva was inoculated orally into control larvae. Some larvae were dissected at 30 min and others at 60 min after inoculation. The midguts were removed and fixed in 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7, for 1 hr at 5°C and then washed six times with 0.05 M phosphate buffer, pH 7.5, for 3 days. Each midgut was cut longitudinally to expose the lumen and washed again with the buffer for 6 hr. The midguts were immersed in the ferritin-antibody conjugate solution for 1 hr at room temperature (ca. 25°C) and then washed with 0.05 M phosphate buffer, pH 7.5, three times with agitation in a vibrator during a period of 3 hr. The specimens were postfixed with 1% 0~0, in 0.1 M phosphate buffer, pH 7, dehydrated in a graded ethanol series, embedded in Araldite, sectioned with a Porter-Blum MT 2 ultramicrotome, stained with saturated aqueous uranyl acetate, and examined with a Philips EM 300 electron microscope at 40 kV. The evaluation of ferritin deposits was determined quantitatively as follows. Areas that showed predominately cross sections of microvilli were selected at random at a low magnification in the electron microscope and photographed at a high magnification. The micrographs, 20.3 x 25.4 cm, were divided vertically by five lines drawn at 3.8-cm intervals, starting from the lefthand margin. Beginning with the upper left-hand corner of the micrograph, microvilli that touched any of these lines were

252

TANADA

selected for counting ferritin deposits. At each position of the midgut (anterior, middle, and posterior) and at each time period (30 and 60 min postfeeding of SF), 150 microvilli were examined for ferritin deposits. The deposits were tallied as “hits” when the ferritin particles touched the unit membrane or the tilamentous coat (11 nm) of the microvillus, and as “nears” when the particles occurred beyond the end of the lilamentous coat plus an additional 10 nm in the lumen. RESULTS

The infectious hemolymph from NPVinfected larvae produced 100% larval infection at dilutions below lop4 (Table 1). The 50% infectious dose was at the dilution of approximately 10m5. The SF inoculated orally did not enhance the NPV inoculated into the larval hemocoel. At the higher dilutions, 10e5 and 10e6, where less than 100% infection occurred, there appeared to be no significant difference in the level of virus infection with and without SF. The enveloped virions were infectious when inoculated orally and intrahemocoelitally, but a much lower virus concentration was needed for the latter route of infection (Table 2). The SF enhanced the infection of the purified enveloped virions of NPV when inoculated into the midgut, but not in the hemocoel. In cross section, the microvillus is ca. 0.15 pm in diameter. It is bounded with a unit plasma membrane, the exterior of which is surrounded by a polysacchariderich coat of filaments, ca. 11 nm in length (C, Fig. 1). Within the microvillus, there is a core of randomly spaced filaments (F, Fig. 1). Electron-dense granules (S, Fig. 1) of various shapes and sizes are scattered on and around the microvillus. The nature of

ET

AL.

these granules is unknown, but they differ from the ferritin particles that are resolved at high magnifications into a tetrad of electron-dense micelles, ca. 1.3 nm apart. The ferritin complex measured, at its widest diameter, about 5.5 nm. The midgut lumina of control larvae had very small amounts of ferritin particles, whereas those of larvae exposed to SF had high numbers of particles (Table 3). Most of the particles were “hits” in close association with or in direct contact with the surface of the microvilli (Fig. 2). This indicated that the SF, which combined with the conjugated antibody, was associated with the surface of the cell membrane. Only a few scattered ferritin particles were found in open areas between the microvilli. The total quantity of ferritin particles observed in the anterior midgut portion was slightly less than that observed in the middle and posterior portions at both 30 and 60 min postinoculation of SF. The quantity of ferritin deposits in the midguts examined 30 and 60 min post-SF treatment was approximately the same. DISCUSSION

The infectious unit of a baculovirus occurs as unenveloped (naked) and as enveloped virions whose envelopes vary in origin. The infectivity of these units varies with the site of infection, the midgut or the hemocoel (Khosaki and Himeno, 1972; Kawarabata, 1974; Dougherty et al., 1975; Summers and Volkman, 1976; Volkman et al., 1976; Volkman and Summers, 1977; Kawarabata and Aratake, 1978). In our study, the SF inoculated orally did not enhance the infectivity of the hemolymph virions which had been inoculated into the larval hemocoel. This confirms the earlier study conducted with the capsules of GVH

FIG. 1. Cross sections of microvilli of midgut cells of the armyworm ferritin-conjugated antiserum in the absence of the synergistic factor. filaments; F, randomly spaced filaments; S, electron-dense granules. FIG. 2. Cross sections of microvilh of midgut cells of the armyworm with the synergistic factor and then treated with the ferritin-conjugated of ferritin particles (arrows) attached and adjacent to the surfaces of

which had been treated with the C, polysaccharide-rich coat of Bar = 100 nm. which had been fed initially antiserum. Note the abundance microvilh. Bar = 100 nm.

ACTION

1 I ,;

SITE

OF

VIRUS

SYNERGISTIC

FACTOR

253

TANADA

254

ET AL.

TABLE NUMBER

3

OF FERRI TIN DEPOSITS OBSERVED WITH THE ELECTRON MICROSCOPE ON THE ANTERIOR, MIDDLE, AND POSTERIOR PORTIONS OF MIDGUTS OF ARMYWORM LARVAE INOCULATED ORALLY WITH

SYNERGISTIC FACTOR AND WITH BUFFER (CONTROL)” Anterior

Middle

Posterior

Treatment

Time (min)

Hits

Near

Hits

Near

Hits

Near

Total

Synergistic factor

30 60

23 25

22 32

48 38

28 36

54 40

34 21

209 192

Buffer (control)

30 60

2 7

2 2

0 0

2 4

4 1

5 3

15 17

fl The figures represent observations made on 150 microvilli at each of three portions of the midgut.

(Tanada and Hukuhara, 197 1). Apparently, the SF is incapable of penetrating through the midgut into the hemocoel or it does not enhance the infectious hemolymph virions within the hemocoel. The infection of baculovirus within the midgut is initiated by the fusion of the enveloped virion to the cell membrane of the microvillus (Harrap, 1970; Kawanishi et al., 1972; Tanada et al., 1975; Granados, 1978). In a preliminary study, Tanada et al. (1975) have reported that the SF, when fed together with NPV to armyworm larvae, increases the number of NPV-enveloped virions attached to cell membranes of microvilli. In our present study, the infectivity of purified enveloped virions, obtained from polyhedra, is also enhanced by SF within the midgut, but enhancement does not occur when SF and enveloped virions are inoculated together into the hemocoel. The enveloped virions alone, however, are infectious in the hemocoel, although we do not know the role played by the envelope in such an infection. The presence of large quantities of conjugated ferritin particles attached to or near the cell membrane indicates that the homologous SF antigen is present in these areas. Its presence on the cell membrane suggests that it acts on the fusion of viral envelope to the cell membrane. This observation supports the previous finding that the SF enhanced the attachment of enveloped virions (Tanada et al., 1975). The quantities of ferritin deposits did not differ

significantly at 30 and 60 min post-SF inoculation. This indicates that the SF is not readily discharged through peristalsis of the digestive tract. In the silkworm, Bombyx mori, and the tent caterpillar, Malacosoma disstria, food passes completely through the digestive tracts within 80 to 100 min (Heimpel and Angus, 1959). Moreover, GVH capsules, when fed 5 days prior to the feeding of NPV, still enhanced the NPV infection (Tanada, 1959). The basis for the enhanced attachment is unknown but it may be associated with the phospholipids that are found in the synergistic factor (Yamamoto and Tanada, 1978a), in the viral envelope (Yamamoto and Tanada, 1978b), and in the host cell membrane. The electrical charges on the surfaces of the viral envelope and the cell membrane may also be involved in attachment (Yamamoto and Tanada, 1978b, 1980). ACKNOWLEDGMENTS We thank Dr. T. Yamamoto for providing samples of the purified synergistic factor and of the enveloped virions. A portion of this study was supported by the National Science Foundation under Grant No. PCM 75-15323 AOI.

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ACTION

SITE OF VIRUS SYNERGISTIC

tion and characterization of a synergistic enzyme from the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta. J. Invertebr. Pathol., 27, 115-124. HARRAP, K. A. 1970. Cell infection by a nuclear polyhedrosis virus. Virology, 42, 311-318. HEIMPEL, A. M., AND ANGUS, T. A. 1959. The site of action of crystalliferous bacteria in Lepidoptera larvae. J. Insect Pathol., 1, 152- 170. KAWAMURA, A., JR. 1969. “Fluorescent Antibody Techniques and their Applications.” University of Tokyo Press, Tokyo. KAWANISHI, C. Y., SUMMERS, M. D., STOLTZ, D. B., AND ARNOTT, H. J. 1972. Entry of an insect virus in vivo by fusion of viral envelope and microvillus membrane. J. Znvertebr. Pathol. 20, 104- 108. KAWARABATA, T. 1974. Highly infectious free virions in the hemolymph of the silkworm (Bombyx mori) infected with a nuclear polyhedrosis virus. J. Invertebr. Pathol.. 24, 196-200. KAWARABATA, T., AND ARATAKE, Y. 1978. Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori. J. Invertebr. Pathol., 31, 329-336. KHOSAKA, T., AND HIMENO, M. 1972. Infectivity of the components of a nuclear polyhedrosis virus of the silkworm, Bombyx mori. J. Invertebr. Pathol., 19, 62-65. SUMMERS, M. D., AND VOLKMAN, L. E. 1976. Comparison of biophysical and morphological properties of occluded and extracellular nonoccluded baculovirus from in vivo and in vitro host system. J. Virol., 17, %2-972. TANADA, Y. 1956. Some factors affecting the susceptibility of the armyworm to virus infections. J. Econ. Entomol.,

49, 52-57.

Y. 1959. Synergism between two viruses of the armyworm, Pseudaletia unipuncta (Haworth)

TANADA,

(Lepidoptera, 215-231.

FACTOR Noctuidae).

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Y., HESS, R. T., AND OMI, E. M. 1975. Invasion of a nuclear polyhedrosis virus in midgut of the armyworm, Pseudaletia unipuncta, and the enhancement of a synergistic enzyme. J. Invertebr. Pathol., Xi, 99- 104. TANADA, Y., HIMENO, M., AND OMI, E. M. 1973. Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm. J. Invertebr. Patho/.. 21, 31-40. TANADA, Y., AND HUKUHARA, T. 1971. Enhanced infection of a nuclear-polyhedrosis virus in larvae of the armyworm, Pseudaletia unipuncta, by a factor in the capsule of a granulosis virus. J. Znvertebr. Patho/., 17, 116- 126. VOLKMAN, L. E., AND SUMMERS, M. D. 1977. Autographa californica nuclear polyhedrosis virus: Comparative infectivity of the occluded, alkaliliberated, and nonoccluded forms. J. Invertebr. Pathol., 30, 102- 103. VOLKMAN, L. E., SUMMERS, M. D., AND HSIEH, C. H. 1976. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: Comparative neutralization, comparative infectivity, and in vitro growth. J. Viral., 19, 820-832. YAMAMOTO, T., AND TANADA, Y. 1978a. Phospholipid, an enhancing component in the synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta. J. Invertebr. Pathol., 31, 48-56. YAMAMOTO, T., AND TANADA, Y. 1978b. Biochemical properties of viral envelopes of insect baculoviruses and their role in infectivity. J. Invertebr. Pathol.. 32, 202-211. YAMAMOTO, T., AND TANADA, Y. 1980. Acylamines enhance the infection of a baculovirus of the arunipuncta (Noctuidae, myworm, Pseudaletia Lepidoptera). J. Invertebr. Pathol. 35, 265-268.

TANADA,