USDA, Agricultural Research Service, Biological Control of Insects Research Laboratory, 1503 S. Providence Road, Columbia, MO 65203, USA
Correspondence
Holly J. R. Popham
pophamh{at}missouri.edu
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ABSTRACT |
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INTRODUCTION |
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The immune response of insects to bacterial or fungal infection and to filarial or parasitoid infestation is well documented (Gillespie et al., 1997; Carton & Nappi, 1997
; Hultmark, 2003
; Kumar et al., 2003
; Leulier et al., 2003
). A suite of antimicrobial peptides, enzymes and metabolites that limit the proliferation of microbes within the host are induced on detection of pathogen-associated biopolymers, and secreted into the haemolymph. In addition, haemocytes present in the haemocoel are recruited to the foci of infection, phagocytosing smaller microbes and encapsulating larger invaders. Acting in concert, these humoral and cellular responses halt microbial infection and clear, or wall off, the resulting debris from the haemocoel by wrapping it in a toxic shroud of cross-linked protein, melanin and melanized haemocytes (Gillespie et al., 1997
; Carton & Nappi, 1997
). Recently, immunosuppression of pestiferous moth larvae by chemical or biological means has revealed tantalizing hints of a cell-mediated antiviral immune response (Washburn et al., 1996
, 2000
). A generalized immunosuppression caused by parasitoid wasp oviposition (and their symbiotic polydnaviruses) prevents the clearance of an otherwise non-fatal baculovirus infection (Washburn et al., 1996
; Coudron et al., 1997
, 1999
; Shelby & Webb, 1999
; Escribano et al., 2000
; Trudeau et al., 2001
).
Antiviral activities from insect haemolymph (Chernysh et al., 2002) or cultured insect cells (Ridel & Brown, 1979
) have been described. Conversely, baculovirus promoting factors have been isolated from larval silkworm plasma (Kanaya & Kobayashi, 2000
). The plasma enzyme phenol oxidase (PO; L-DOPA : oxygen oxidoreductase; EC 1.14.18.1) of the tobacco budworm, Heliothis virescens, exhibits antiviral activity against several vertebrate viruses (Ourth & Renis, 1993
). Inhibition of the host melanization response by parasitoids, filarial parasites and bacteria, as well as by entomopathogenic fungi, is abetted via reduced PO activity (Shelby & Webb, 1999
; Shelby et al., 2000
). We examined the possibility of Heliothis virescens haemolymph having antiviral activity against an insect virus. Our results extend and confirm the results of Ourth & Renis (1993)
by demonstrating that brief in vitro incubation of Helicoverpa zea single capsid nucleopolyhedrovirus (HzSNPV) with plasma from Heliothis virescens reduces the infectivity of the virus.
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METHODS |
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Collection and processing of plasma.
Early fourth-instar larvae were surface-sterilized in ethanol, rinsed with sterile water and anaesthetized on ice before bleeding. Haemolymph was gently extruded from an anterior proleg through a small puncture wound made with a sterile 26-gauge needle, and collected directly into a chilled 1·5 ml microcentrifuge tube containing ice-cold, sterile PBS (Shelby et al., 2000). Haemolymph was adjusted to a final dilution of 1 : 10 by addition of cold PBS, after which haemocytes were removed by microcentrifugation at 5900 g for 3 min. The plasma supernatant was sterilized by centrifugation at 3200 g for 3 min through a 0·65 µm Millipore Ultrafree-MC centrifugal filter. All plasma samples were collected during the morning.
Plasma in vitro virucidal assay.
Heliothis virescens plasma dilutions were combined with HzSNPV at a ratio of 3 : 1 (v/v), gently mixed and incubated at 20 °C for 1 h. PBS was used as a control in the absence of plasma. Virus titres of these incubations were determined by end-point dilution assay (Summers & Smith, 1987; Slavicek et al., 2001
). HzAM1 cells were seeded at 5x104 cells ml1 in 96-well plates (BD Falcon) and allowed to attach for 1 h. The cells were infected with dilutions of virus/plasma or virus/PBS at dilutions of 101 to 106 and plates were incubated for 1 week at 28 °C. The plate wells were then scored positive for virus infection if polyhedra were visible within two or more cells, or negative for viral infection, and the results were used to calculate the virus titre as the TCID50 (ml inoculum)1 (Slavicek et al., 2001
). Statistical comparisons were done with SigmaStat 3.0 or SigmaPlot 8.0 (SPSS). The StudentNewmanKeuls procedure was used for multiple comparisons when significant variation of the means was found when one-way ANOVA was performed (P<0·001).
Cell viability assay.
Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) dye reduction assay in which viable cells convert MTS into a coloured formazan product (A492) (Cory et al., 1991). HzAM1 cells (5x104 ml1) were plated in 96-well microtitre plates and incubated with 1 : 10 serial dilutions of plasma and virus for 7 days. Following the manufacturer's instructions, 20 µl MTS (CellTitre 96 AQueous One; Promega) was added to 100 µl of culture medium, incubated for 14 h at 20 °C and the A492 was determined with a Perkin-Elmer HTS 7000 Plus plate reader. Results reported are means of eight wells±SD, and each experiment was repeated at least three times.
Plasma treatments.
Heat-inactivation experiments were done by incubation in a water-bath at 65 or 55 °C for 20 min. Samples were cooled before addition of virus. Plasma samples stored at 85 °C for 1 week were thawed on ice before incubation with HzSNPV.
Plasma was subjected to limited inactivating proteolysis with proteinase K by incubating with proteinase K conjugated to polystyrene beads (#P0803; Sigma). Polystyrene beads (400 µl) were combined with an equal volume of 1 : 10-diluted plasma for 30 min at 20 °C with occasional agitation. The mixture was then filter-sterilized by centrifugation at 4000 g for 3 min. A cocktail of protease inhibitors with broad specificity for inhibition of serine, cysteine, aspartic and metalloproteases (#P2714; Sigma) was added at a final concentration of 10 µg ml1 during collection of plasma.
In some experiments PO activation during plasma collection and subsequent incubation was inhibited by addition of the copper chelator 1-phenyl-2-thiourea (PTU) to plasma as larvae were being bled into PBS. The final concentration of PTU was 100 µM in 1 : 10-diluted plasma. To activate prophenol oxidase fully, freshly collected plasma was incubated with 1 µg lipopolysaccharide (LPS) ml1 for 30 min at 20 °C to activate the melanization cascade (Kanost et al., 2001). All experiments were repeated at least three times and representative experiments are presented.
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RESULTS |
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DISCUSSION |
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As in other insects, microbial challenge of Heliothis virescens larvae induces expression of a suite of antibacterial and antifungal proteins such as cecropins A, B and C and attacins (Ourth et al., 1994), lysozyme (Lockey & Ourth, 1996
; Shelby et al., 1998
; Chung & Ourth, 2002
), heliocin, heliomicin (Lamberty et al., 1999
), virescein and viresin (Chung & Ourth, 2000
). Insect prophenol oxidase exists in plasma as a zymogen, in multimeric complexes with other proteins, and is secreted by the fat body, haemocytes, cuticular epidermis and other tissues (Asano & Ashida, 2001
). It is activated to PO by the presence of fungal 1,3-
-glucans, LPS from Gram-negative bacteria and peptidoglycans of Gram-positive bacteria (Kanost et al., 2001
). The plasma PO of Heliothis virescens exhibits antiviral activity against several vertebrate viruses in vitro (herpes simplex virus types 1 and 2, vesicular stomatitis virus, human parainfluenzavirus 3, coxsackievirus B3 and Sindbis virus) (Ourth & Renis, 1993
). Of these, vesicular stomatitis virus and Sindbis virus were the only viruses tested which are known to infect insects or insect cells (Lewis et al., 1999
). Activity against known insect viruses such as baculoviruses was not tested, and is the subject of this report. The antiviral activity was present constitutively in plasma, i.e. immunization by bacterial injection did not increase activity over naïve controls. The authors attributed the observed antiviral activity in Heliothis virescens plasma to PO because: (i) it was inhibited by PTU and addition of EDTA/citrate to plasma; (ii) the antiviral effect could be restored to a plasma chromatographic fraction containing PO activity; (iii) by addition of known PO substrates; and (iv) the antiviral effect could be mimicked in vitro by incubation with mushroom tyrosinase and the same substrates (Ourth & Renis, 1993
). With a similar assay we have found that, although HzSNPV was substantially inactivated by Heliothis virescens plasma, the level of inhibition was much lower than that seen against vertebrate viruses, for which Heliothis virescens is not a host.
The mode of action against HzSNPV appears to be direct, as it occurs in vitro, before addition to HzAM1 cells. Addition of plasma and virus directly to HzAM1 cells, bypassing the 20 °C in vitro incubation, did not yield significant differences in the TCID50. Pre-incubation of HzAM1 cells for 2 h with 1 : 10-diluted plasma before addition of HzSNPV did not affect the subsequent course of infection; TCID50 values were not affected. This would seem to militate against mechanisms whereby the uncharacterized factor interferes with the infective process by blocking a required receptor, or by activating HzAM1 cells to render them more resistant to initial infection. The known inhibitory action of PTU, a copper chelator, against the Heliothis virescens plasma PO, provides a strong argument for involvement of this enzyme in the virucidal activity against HzSNPV detailed above.
High molecular mass melanogenic and sclerotinogenic multimeric protein complexes (multimers of prophenol oxidase, dopachrome tautomerase, interleukin 1-like molecule and other enzymes) are present at high concentration in insect plasma (Beck et al., 1996), and are thought to participate in the recognition and opsinization of invading pathogens, and in the recruitment of haemocytes, which phagocytose smaller pathogens (Marmaras et al., 1996
; Sugumaran, 2002
). The density-dependent prophylaxis against a wide variety of entomopathogens observed in lepidopteran larvae is directly correlated with elevated cuticular and plasma melanizing capability, i.e. PO activity (Reeson et al., 1998
; Wilson et al., 2001
). Conversely, reduction of plasma melanizing activity, by targeting prophenol oxidase expression in the mosquito Armigeres subalbatus with a Sindbis virus encoding an antisense transcript, eliminated melanization of Dirofilaria immitis microfilaria (Shiao et al., 2001
). Generation of cytotoxic free radicals (e.g. quinones, quinone methides, semiquinones, superoxide, hydrogen peroxide, nitric oxide, peroxynitrite) in the haemolymph of insects by these soluble enzymic activities and by LPS-stimulated haemocytes is a well-documented response to microbial infection, and to filarial and parasitoid infestation (Luckhart et al., 1998
; Nappi & Ottaviani, 2000
; Kumar et al., 2003
; Leulier et al., 2003
). The possible virucidal activity of these free radicals in infected insects has not yet been adequately explored.
In conclusion, we have documented the presence of an antiviral activity, present in the plasma of susceptible larvae, which is active against baculoviruses in vitro. Inhibitor and substrate studies indicate that the virucidal activity is coincident with activity of the plasma enzyme PO. Isolation and further characterization of this virucidal activity will be the subject of a future report. The mechanism by which this factor can distinguish the invading virus from host proteins and tissues residing within the haemocoel remains an extremely intriguing question, which we intend to pursue, as is the possibility that this activity may be involved in vector competence of arthropods that transmit viruses to vertebrates or plants.
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ACKNOWLEDGEMENTS |
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Received 8 January 2004;
accepted 6 May 2004.
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