Antiviral response in insects?

JOURNAL

OF INVERTEBRATE

PATHOLOGY

29, 44-49 (1977)

Antiviral J. KALMAKOFF,' Department

of Microbiology

Response in Insects?

B. R. G. WILLIAMS,'

and Virus Research University of Otago,

Unit, Medical Dunedin, New

AND F. J. AUSTIN' Research Zealand

Council

of New

Zealand,

Received December 30, 1975 The induction of an antiviral response in cultured Aedes aegypti cells was compared with that in duck embryo fibroblasts using 32P-labeled double-stranded RNA (dsRNA) from reovirus and poly(rI-rC). Although A. aegypti cells could incorporate a comparable amount of dsRNA @OS), no antiviral response was observed. Further studies using poly(rI-rC) under conditions shown to be optimal for vertebrate cells failed to give positive results. An attempt in Bombyx mori larvae to measure a cellular response to foreign material (a coliphage) injected into the hemocoel resulted in the persistence of the virus for at least 8 days.

INTRODUCTION

An aspect of the use of insect viruses for biological control programs that has received little serious consideration is the defense mechanism of insects to virus diseases. Do insects have any immune or antiviral responses and, if so, are there any means by which the susceptibility of insects to disease can be increased? There have been reports that latent diseases become activated in insects subjected to stress conditions such as cold shock, starvation, or treatment with chemicals (Matsubara, 1968; Watanabe, 1971). The mechanism of activation is not known, but presumably, if the viruses were being “carried” but not active, this would indicate that a defense system is operating which fails under stress conditions. Some suggestion that this occurs is supported by finding viral DNA in apparently healthyspodoptera littoralis larvae (Kislev et al., 1971). The phenomenon of the “gut barrier” may be very significant in insect resistance, and it is possible that the defensive mechanism in insects is designed to prevent the penetration of virus particles into the hemocoel (Tinsley, 1975). The difference in dosage rates necessary to infect per OS or 1 Department of Microbiology. 2 Virus Research Unit.

intrahemocoelically is often given as circumstantial evidence for this view. This difference, however, may be due to destruction of the viruses by digestive enzymes in the insect gut, or the gut cells may represent a barrier of nonsusceptible tissue. It has been shown that insects do not possess immunoglobulins normally associated with the immune response in higher animals (Hildemann, 1974). It is possible that there is a cellular response to disease in the hemocoel, the hemocytes being the equivalent of the macrophage and lymphocyte system in higher animals (Anderson, 1975). However, the evidence to support this view is not compelling. Interferon, however, may represent a more primitive response to virus infection since it displays a spectrum of action against a wide variety of viruses and is probably present in all vertebrates. Attempts have been made to demonstrate an interferonlike response in cultured mosquito cells with negative results (Murray and Morahan, 1973; Kascak and Lyons, 1974). It has been shown that double-stranded ribopolymers (dsRNA), particularly the synthetic polyribonucleotide poly(rI-rC), are potent inducers of interferon (Colby and Chamberlin, 1969). Recent experiments using poly(t-I-rC) linked to an insoluble support have shown that it is probable that dsRNA must 44

Copyright All rights

0 1977 by Academic Press. Inc. of reproduction in any form reserved.

ANTIVIRAL

RESPONSE

enter the cell as a prerequisite step in the induction of an antiviral response (Hutchison and Merigan, 1975). It is possible that the lack of interferon induction in insect cells using various inducers is due to lack of incorporation of dsRNA into the cells. This paper describes attempts to elicit an interferon-like response in Aedes aegypti cells under conditions where dsRNA is incorporated into cells and also under conditions shown to be optimal for vertebrate cell culture systems using poly(rI-rC) (Kalmakoff and Austin, 1973). Some preliminary experiments on the “clearance” of a bacterial virus from Bombyx mori are also presented. MATERIALS Culturing

AND METHODS

of mosquito

and duck embryo

cells. Monolayers of the A. aegypti cell line of Singh (1967) and A. aegypti of Peleg (1968) were cultured in 30-ml Falcon plastic cell culture bottles or in 100 x lo-mm glass test tubes. Cells were trypsinized with Rinaldini’s solution and diluted to lo5 cells/ml with M-M medium (Mitsuhashi and Maramorosch, 1964) and dispensed in 4-ml volumes into Falcon bottles or l-ml volumes into test tubes. Cultures were incubated at 28°C and were used 3-4 days after trypsinization. Primary duck embryo cell cultures were prepared as previously described by Kalmakoff and Austin (1973). Glass Petri dishes (60 mm in diameter) or glass test tubes were seeded with 3 x lo6 cells/ml in growth medium. After 3 days of incubation at 37”C, the cells formed a confluent monolayer and were used for viral interference or virus yield assays. Viral

resistance

and virus yield assays.

To induce antiviral resistance, the cells were washed twice with growth medium, and then various amounts of DEAE-dextran (Pharmacia Fine Chemicals) and purified 32P-labeled dsRNA from reovirus (from A. R. Bellamy) or poly(rI-rC) were added. Poly(rI-rC) was purchased from P-L Biochemicals, Milwaukee, Wisconsin, and dis-

IN INSECTS?

45

solved in buffer containing 0.14 M NaCl, 10 mM phosphate, 27 mM KCl, 0.9 mM CaCl,, and 0.5 mM MgClz * 6Hz0, pH 7.4, at 45°C for 3 hr, then stored at -20°C until used. After the induction period (either 3 or 24 hr), the cultures were washed twice with growth medium to remove the inducer and challenged with 2 x 10“ plaqueforming units (PFU) of Whataroa virus or Semliki Forest virus, both group A arboviruses. After 40 min at 37” C, the unadsorbed virus was removed and the cultures were washed twice with growth medium. After 18 or 48 hr, the medium was collected and the virus yield was determined by infecting primary duck embryo cell monolayers and counting the plaques formed (Kalmakoff and Austin, 1973) . Uptake of 32P-labeEed dsRNA by cells. 32Plabeled dsRNA from reovirus was added to cells cultured in l-ml volumes in test tubes and incubated for 24 hr at 28” or 37°C for A. aegypti or duck embryo cells, respectively. The cells were then washed three times with ice-cold 0.1 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl. One milliliter of 5% trichloracetic acid (TCA) was added, and the cells were placed on ice for 30 min. The supernatant solution containing the cold TCA-soluble fraction (degradation products of dsRNA) was removed and the radioactivity determined. The cells were washed three times with cold 5% TCA. One milliliter of 10% TCA was added, and the tubes were incubated at 65°C for 1 hr. This leads to solubilization of all macromolecular dsRNA (cellassociated dsRNA) for counting of radioactivity. The amount of dsRNA incorporated was calculated by reference to the amount initially added. Attempts to detect interferon from persistently infected A. aegypti cells. Monolayers of A. aegypti cells in Falcon flasks

were infected with Whataroa virus and then cultured for 131 days; when monolayers became confluent in 4-6 days, they were trypsinized and reseeded. The culture fluid when assayed on duck embryo monolayers

46

KALMAKOFF.

WILLIAMS

contained about lo4 PFU/ml. This virus was inactivated and removed from the culture medium by treating with heat, 56°C for 1 hr, followed by ultracentrifugation at 100,OOOg for 60 min. Normal monolayers of A. aegypti cells were washed with growth medium and then treated with the fluid from the persistently infected culture for 24 hr at 28°C. After removing the inducing fluid, the cells were washed with growth medium and then challenged with virus and assayed for virus yield as described above. Uptake of Hd virus by A. aegypti cells. Hd, a male-specific icosahedral coliphage, was grown in Escherichia coli AB259 (an Hfr strain) in L-medium and purified as previously described (Bilimoria and Kalmakoff, 1971). The uptake of Hd virus was studied by incubating A. aegypti cells with virus containing various concentrations of DEAE-dextran for 3 hr at 28°C. The virus remaining unadsorbed was measured by plating the cell culture medium on E. coli AB259 cells using the agar overlay method and counting the plaques formed (Overby et al., 1966). Uptake of Hd virus by B. mori larvae. Fourth- or fifth-instar B. mori larvae (strain C-124) reared on fresh mulberry leaves were injected via the last proleg with 10 ~1 of Hd virus (lo6 PFU) using a microsyringe. At various times after inoculation 10 larvae were removed, frozen, and stored at -20°C. Each larva was ground with a mortar and pestle and sterile sand in 5 ml of phosphate-buffered saline (PBS). The larval debris and sand were removed by centrifugation at 2000 rpm in a bench-top centrifuge. Virus was assayed by plating on E. coli AB259 cells as described above. RESULTS AND DISCUSSION

Since it has been shown that it is important that the dsRNA must enter the cell to induce an antiviral activity, the uptake of reovirus dsRNA was studied.

AND AUSTIN

We have previously shown that DEAEdextran is important in the uptake of dsRNA for the induction of viral resistance in primary duck embryo cells (Kalmakoff and Austin, 1973). With 10 pg/ml of DEAEdextran present in the induction medium, the duck embryo cells incorporated 7% of the added dsRNA, whereas in the absence of DEAE-dextran only 12% became cell associated (Table 1). The A. aegypti cells, however, incorporated only 23% of the added dsRNA under these conditions. When higher concentrations of DEAEdextran were added with a constant amount of dsRNA (28 pg/ml), it was possible to get 80% of the dsRNA incorporated into the cells. However, on challenge with Whataroa virus, there was no significant reduction in virus yield when compared to the control without dsRNA (Table 1). Duck embryo cells, on the other hand, treated under similar conditions but using lower DEAE-dextran levels gave almost a 4.0-log reduction in virus yield. It would appear that, although the A. aegypti cells could incorporate an amount of dsRNA comparable to that incorporated by duck embryo cells, 80 and 79%, respectively, there was no interferon or viral resistance system operative. The role that DEAE-dextran plays in potentiating the induction of interferon by dsRNA is not known; however, it is thought that it increases the efficiency of uptake of macromolecules by cells or protects the dsRNA from degradation by nucleases. The part played by DEAE-dextran was investigated further by studying the uptake of Hd virus, an icosahedral virus 25 nm in diameter with a molecular weight of 3.6 x lo6 daltons (Overby et al., 1966). When 10 pg/ml of DEAE-dextran was used, 98% of the virus added was removed from the cell culture medium (Table 2). The mechanism of uptake is probably by pinocytosis, indicating that the large pieces of reovirus dsRNA (MW = 2.5 x lo6 daltons) can also be incorporated by a similar mechanism. This would suggest that DEAE-dextran

ANTIVIRAL

TABLE THE EFFECT

OF

Cell culture Aedes

dsRNA INCORPORATED

DEAEdextran (PLP)

2.8 -

-

aegypti

1

BY CELLS ON VIRUS YIELDS

dsRNA (/a)

47

RESPONSE IN INSECT?

dsRNA incorporateda (%)

WHEN

CHALLENGED

BY WHATAROA

VIRUS

Yield b (PFUlml)

Log PFU

Alog PFU

19 -

8.0 x lo4 4.0 x 105

4.9 5.6

0.7

A. aegypti

2.8

10 10

23 -

ndc nd

A. aegypti

2.8

30 30

60 -

8.0 x lo4 1.2 x 105

4.9 5.1

0.2

A. aegypti

2.8 -

100 100

80 -

8.0 x 103 1.0 x 104

3.9 4.0

0.1

Duck embryo

2.8 -

-

12 -

1.6 x 10’ 1.3 x 10’

7.2 7.1

0.1

Duck embryo

2.8 -

79

2.0 x 103 1.6 x 10’

3.3 7.2

3.9

10 10

-

a The percentage of 32P-labeled dsRNA incorporated by the cells after 24 hr. * Virus yield 48 hr after challenging the cells with 2.5 x lo3 PFU of virus. c Not done.

potentiates the effect of dsRNA by increasing the uptake of macromolecules by cells rather than by protecting the dsRNA from nuclease degradation. One of the most potent inducers of interferon in vertebrate cells has been found to be the synthetic dsRNA polyinosinic-polycytidylic acid (poly(rI-rC)). We have previously investigated the optimum conditions for inducing antiviral activity with this dsRNA using chick embryo fibroblasts TABLE THE

2

EFFECT OF DEAE-DEXTRAN ON THE UPTAKE OF Hd VIRUS BY Aedes aegypti CELLS

DEAE-dextran added (PLP) 10 30 100

Recovered” (PFU/ml) 5 8 4 4

x 106 x 104 x 104 x 103

Log PFU

PFU (%I

6.7 4.9 4.6 3.6

100 1.6 0.8 0.08

n The virus was incubated for 3 hr at 28°C with aegypti cells, then the virus remaining in the cell culture medium was assayed by plating on E. coli AB259 cells.

Aedes

(Kalmakoff and Austin, 1973). It was found that a ratio of poly(rI-rC)/DEAE-dextran of 0.1 was the optimum. Using this optimum ratio, A. aegypti (Peleg) cells were tested for an antiviral response. The results in Table 3 indicate that no antiviral activity was found since the virus yields from cultures induced for 3 hr were similar to the controls. From studies of persistently infected vertebrate cells it has been found that interferon is a mechanism whereby a virus infection can be maintained (Field et al., 1968). Cell culture fluid from A. aegypti cells which were persistently infected with Whataroa virus was tested for an interferon-like activity by adding the fluid to normal A. aegypti cells which were then challenged with Whataroa virus. There was no evidence of an interferon like activity being present in the persistently infected cells (Table 4). The results were similar to those reported by Kascack and Lyons (1974) and Davey and Dalgarno (1974). The above studies which have been carried out using cells in vitro, may not be

48

KALMAKOFF.

WILLIAMS

AND AUSTIN

TABLE 3 THE EFFECT OF POLY(rI-rC)/DEAE-DEXTRAN ON VIRUS YIELDS IN Aedes aegypti CELLS WHEN CHALLENGEDBY SEMLIKI FOREST VIRUS I

8- I

Treatment Poly(rI-rC) (wdml)

DEAEdextran Wml)

1 10 3 30 10 100 30 300 Control PBS

Virus yield” (PFU/ml) 1.4 7.6 4.5 7.9 9.0

x x x x x

105 104 105 104 104

(log PFU) 5.15 4.88 5.65 4.9 4.95

n The cells were incubated with the inducer for 3 hr at 28”C, washed with PBS, and challenged with Semliki Forest virus.

relevant to the in vivo situation. A difficulty in dealing with viruses in whole insects is the problem of assaying virus yields from insects since no quantitative method is readily available. However, recent developments in plaquing insect viruses (Hink and Vail, 1973) and the use of radioimmunoassay (Bilimoria et al., 1974) may overcome these difficulties. It has been shown that insect hemocytes are efficient in removing bacteria, fungi, and nematodes from the hemocoel (Salt, 1970), and recently Ratcliff and Rowley (1975) demonstrated in vitro phagocytosis of bacteria by hemocytes of several species of insects. InB. mori we attempted to measTABLE 4 TEST FOR INTERFERON IN PERSISTENTLY INFECTED Aedes aegypfi CELLS Virus yield Treatment Cells treated with fluid from infected cultures Cells treated with fluid from uninfected cultures No treatment

(PFU/ml)

(log PFU)

5.5 x 106U

6.74

1.0 x lo6 1.2 x 108

6.0 6.08

n Mean from two experiments using different persistently infected cultures.

Input

10

2n

30 40 50 hours post lrmutatlon

60

70

@I

FIG. 1. The clearance of Hd virus from inoculated Bombyx mori larvae. The virus at lo6 PFU was inoculated into the hemocel, then, at each time period, larvae were removed and assayed for virus. The vertical bars represent 1 SD.

ure a cellular response to injection of foreign material into the hemocoel. Larvae were injected with 10 ~1 of Hd virus and then assayed for viable virus by plating on E. co/i AB259. It can be seen (Fig. 1) that, after an initial drop in virus titer, due to freezing of the samples, there was no significant decrease in virus present in the hemocoel after 72 hr. If the insect recognized this material as “foreign,” then the virus should have been cleared by phagocytosis. It is remotely possible that the virus, although being cleared from the hemocoel, was also multiplying in the bacteria of the insect gut microbiota such that an equilibrium between clearance and multiplication was established. Subsequent investigation showed that there were no E. co/i Hfr strains present in the microbiota of the insect and that the virus reisolated was Hd virus. Another experiment carried out for 8 days gave similar results. In view of these negative findings it is possible that either antigenic foreignness is not recognized by B. mori larvae or they do not recognize a bacterial virus as foreign since it is not an insect pathogen. Studies using inactivated insect viruses may resolve this problem. Since insects are a very successful class of animals, it seems likely that they have

ANTIVIRAL

49

RESPONSE IN INSECTS?

solved the problem of virus infections during their evolution. It has been shown that a primitive cell-mediated immunity exists in echinoderms and annelids (Hildemann, 1974). It is, therefore, likely that insects also possess a similar immune response; however, whether they have an antiviral defense system still remains to be demonstrated. ACKNOWLEDGMENTS This study was supported in part by the Medical Research Council of New Zealand. We gratefuly acknowledge the gift of 32P-labeled reovirus RNA from A. R. Bellamy and the technical assistance of Gavin Murray.

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lack of antiviral effect of (polyinosinic acid):(polycytidylic acid) when attached to insoluble supports. J. Gen. Virol., 27, 403-407. KALMAKOFF, J., AND AUSTIN, F. J. 1973. Induction of viral interference: Effects of poly rI-rC and diethylaminoethyl dextran on the activity of the antiviral protein. Infect. Zmmun. 8, 63-67. KASCSAK, R. J., AND LYONS, M. J. 1974. Attempts to demonstrate the interferon defense mechanism in cultured mosquito cells. Arch. Gesamte Virusforsch., 45, 148-154. KISLEV, N., EPELMAN, M., AND HARPAZ, I. 1971. Nuclear polyhedrosis viral DNA: Characterization and comparison to host DNA. J. Znvertebr. Pathol., 17, 199-202. MATSUBARA, F. 1968. Studies on the diseases of the aseptically reared silkworms, Bombyx mori (L) V. Induction of the nuclear polyhedrosis by the feeding of certain chemicals to the silkworm after transfer from aseptic to normal rearing conditions. J. Sericult. Sci. Japan., 37, 137-143. MITSUHASHI, J., AND MARAMOROSCH, K. l%4. Leafhopper tissue culture: Embryonic nymphal and imaginal tissues from aseptic insects. Contrib. Boyce Thompson

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22, 435-460.

MORAHAN, P. S. 1973. Studies of interferon production in Aedes albopticus mosquito cells. Proc. Sot. Exp. Biol. Med., 142, 1 l15. OVERBY, L. R., BARLOW, G. H., Dot, R. H., JACOB, M., AND SPIEGELMAN, S. 1966. Comparison of two serologically distinct ribonucleic acid bacteriophages. 1. Properties of the viral particles. J. Bacterial., 91, 442-448. PELEG, J. 1968. Growth of arboviruses in monolayers from subcultured mosquito embryo cells. Virology, 35, 617-619. RATCLIFF, N. A., AND ROWLEY, A. F. 1975. Cellular defense reactions of insect hemocytes in vitro: Phagocytosis in a new suspension culture system. MURRAY,

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SALT, G. 1970. “The Cellular Defense Reactions of Insects.” Cambridge Monographs in Experimental Biology No. 16, Cambridge University Press, London and New York. SINGH, K. R. P. 1967. Cell cultures derived from larvae of Aedes albopticus (Skuse) and Aedes aegypti (L). Cur. Sci., 36, 506-508. TINSLEY. T. W. 1975. Factors affecting virus infection ofinsect gut tissue. In “Invertebrate Immunity” (K. Maramorosch and R. E. Shope, eds.) pp. 5563. Academic Press, New York. WATANABE. H. 1971. Resistance of the silkworm to cytoplasmic polyhedrosis virus. In “The Cytoplasmic Polyhedrosis Virus of the Silkworm” (H. Amga and Y. Tanada, eds.), pp. 169-184. University of Tokyo Press, Tokyo.