1 Department of Microbiology, Oregon State University, 220 Nash Hall, Corvallis, OR 97331-3804, USA
2 Hawaii Institute of Marine Biology, Kaneohe, HI 96744, USA
Correspondence
Douglas Leisy
leisyd{at}onid.orst.edu
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ABSTRACT |
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INTRODUCTION |
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Baculoviruses, in particular Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV), are widely used for overexpression of cloned genes (O'Reilly et al., 1992). In this system, the gene encoding polyhedrin (the protein comprising the polyhedron matrix in which the occluded viruses are imbedded) is usually replaced with a gene of interest and the very high transcriptional activity conferred by the polyhedrin promoter is exploited for overexpression of the selected gene. Because of the high levels of expression and the fidelity of protein processing, this system is frequently favoured over other eukaryotic expression systems.
In another recent application of baculoviruses, several investigators have reported that AcMNPV is capable of transducing genes into a variety of mammalian cell types (reviewed by Kost & Condreay, 2002). For these investigations, recombinant AcMNPV constructs have been produced that carry expression cassettes consisting of a reporter gene linked either to the human cytomegalovirus (CMV) immediate-early (IE) promoter/enhancer or to the CAG promoter (consisting of the CMV IE enhancer, the chicken
-actin promoter and the rabbit
-globin polyadenylation signal) (Niwa et al., 1991
). With these constructs, it was found that a wide variety of mammalian cell types were capable of being transduced, including a number of human, monkey, porcine and rat cell lines (Condreay et al., 1999
; Shoji et al., 1997
).
Although the ability of baculoviruses to transduce genes into mammalian cells is well established, investigations on their application to other vertebrates have been limited. Because fish are subjected to a variety of virus diseases, the development of efficient methods to prevent these infections is of great importance. Therefore we have initiated detailed experiments to determine whether baculovirus vectors are capable of delivering genetic vaccines to fish. In this report, we demonstrate that baculovirus vectors are effective at transferring a lacZ reporter gene under the control of the CAG regulatory elements into several different types of cultured fish cells. One cell line, epithelioma papulosum cyprini (EPC) (Fijan et al., 1983), derived from carp, was selected for further studies and the effects of m.o.i., temperature and treatment with a histone deacetylase inhibitor (sodium butyrate) were investigated. Expression from a recombinant baculovirus that displays a heterologous viral fusion protein on the virion surface was also examined.
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METHODS |
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Cell lines tested.
The fish cell lines tested for transduction with recombinant baculoviruses were: Tilapia ovary (TO-2) (Chen et al., 1983); epithelioma papulosum cyprini (EPC) cells (Fijan et al., 1983
); rainbow trout gonad (RTG-2) cells (Wolf & Quimby, 1962
); Chum salmon (Oncorhynchus keta) heart fibroblast (CHH-1) cells (ATCC CRL-1680; Lannan et al., 1984
); and chinook salmon embryo (CHSE-214) cells (Lannan et al., 1984
). All cells were maintained in complete MEM-5, which consisted of autoclaved minimal essential medium (GibcoBRL) supplemented with 5 % foetal bovine serum (FBS; Intergen) and 2 mM L-glutamine (GibcoBRL).
Infections and -galactosidase assays.
The high-sensitivity -galactosidase assays were performed by incubating 1x105 cells per well in 24-well tissue culture plates with 100 µl of complete MEM-5 culture medium containing Ac-CAlacZ or AcVSVG-CAlacZ baculovirus at the indicated m.o.i.s. At 1 h post-treatment, 1 ml MEM-5 was added to each well, with or without the addition of sodium butyrate (Sigma), as indicated. At the indicated times, cells were treated with lysis buffer [0·25 M Tris, pH 7·4, 0·25 % (v/v) NP-40, 2·5 mM EDTA] and assayed for
-galactosidase activity using a high-sensitivity
-galactosidase assay kit (Stratagene). Each data-point represents a single
-galactosidase activity determination divided by the protein concentration of the extract. The protein concentration was determined from the average of three assays for each extract, using the DC protein assay (Bio-Rad).
For in situ -galactosidase staining assays, 2x104 cells per well in 96-well plates were incubated with 30 µl of complete MEM-5 culture medium containing Ac-CAlacZ or AcVSVG-CAlacZ baculovirus at the indicated m.o.i.s, with or without the addition of sodium butyrate (Sigma), as indicated. At 1 h post-treatment, 100 µl MEM-5 was added to each well. At the indicated times, cells were stained using an in situ
-galactosidase assay kit (Stratagene).
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RESULTS |
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DISCUSSION |
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The EPC cell line was selected for further study. Dose-response kinetics were examined, both by counting the number of cells expressing -galactosidase activity and by measuring the amount of
-galactosidase activity in lysates from infected cells. At an m.o.i. of 1·6, we observed, on average, only one
-galactosidase-expressing cell per 2x104 cells. Between m.o.i.s of 1·6 and 6·3,
-galactosidase expression rose at a higher rate than the input dosage. Between m.o.i.s of 6 and 25, the number of
-galactosidase-expressing cells was linear with respect to the dose of virus. At m.o.i.s higher than 25, expression levels rose at a rate slightly lower than the increase in dosage level. These results are not in strict agreement with dose-response experiments performed with mammalian cells, which showed a linear relationship with m.o.i., with no saturation occurring even with an m.o.i. as high as 800 (Duisit et al., 1999
).
The addition of sodium butyrate to the culture medium of baculovirus-infected EPC cells resulted in a 5- to 6-fold elevation of -galactosidase activity. Similar results have been reported with mammalian cells (Condreay et al., 1999
). Sodium butyrate maintains histones in an acetylated form by inhibiting histone deacetylase. Addition of sodium butyrate to cells transfected with plasmids using liposomes showed only small or negligible increases in reporter gene activity (data not shown). In contrast to purified plasmids that consist of naked DNA, baculovirus virion DNA most likely has a nucleosome-like structure (Wilson & Miller, 1986
). These data suggest that sodium butyrate may act on virion DNA in a manner similar to its effects on cellular DNA.
We found that reporter gene expression levels in baculovirus-infected EPC cells were much higher at 29 °C than at lower temperatures. Temperature-shift experiments demonstrated that temperatures in the range tested (1729 °C) had no affect on the ability of the cells to adsorb virus, but did affect the level of gene expression after adsorption. We have now tested several different promoters via liposome-mediated transfection of plasmid DNA and found that, in comparison with other promoters tested, the CAG regulatory cassette is particularly temperature-sensitive (data not shown). Since the CAG control elements were used in all of our baculovirus constructs, the observed temperature effects are probably due to reduced transcription of the reporter gene at the lower temperatures.
In AcMNPV, entry into insect cells is mediated by specific interactions between the virion envelope protein gp64 and host receptors (Hefferon et al., 1999). Recent studies suggest, however, that baculovirus binding to mammalian cells may occur through non-specific electrostatic interactions between the viral and cell membranes (Duisit et al., 1999
). Although gp64 may not be important for initial binding and internalization into endosomes, it is probably involved in fusion of the virion envelope to the endosomal membrane, thereby facilitating the release of the nucleocapsid into the cell (Blissard & Wenz, 1992
). A recombinant baculovirus that contained about 1·5 times the normal amount of gp64 on the virion surface exhibited 10- to 100-fold more reporter gene expression in a variety of mammalian cells compared with similar viruses with a normal amount of gp64 (Barsoum et al., 1997
; Tani et al., 2001
). Recombinant baculoviruses that contain heterologous viral envelope glycoproteins, such as the VSV G protein or the mouse hepatitis virus S protein, have also been reported to elevate levels of foreign gene expression in mammalian cells (Tani et al., 2001
). These studies suggest that these proteins may mediate binding to specific receptors on the mammalian cells or release of the virions from endosomes. Using a VSV G-containing baculovirus (AcVSVG-CAlacZ) and the EPC fish cell line, we observed 15-fold elevated levels of
-galactosidase expression, similar to that observed with mammalian cells (Barsoum et al., 1997
).
Recent studies have demonstrated superior efficacy of DNA-based vaccines for immunization of fish compared with more traditional vaccines, such as inactivated or attenuated vaccines. For instance, injection of rainbow trout with a plasmid construct containing the infectious hematopoietic necrosis virus (IHNV) G protein gene under the control of the CMV promoter provided excellent protection against IHNV in viral challenge assays (Anderson et al., 1996). However, the practical application of such DNA-based vaccines is limited because they have only been found to be effective when delivered by injection, gene gun, scarification (Corbeil et al., 2000
), or by the application of short-pulse ultrasound (Fernandez-Alonso et al., 2001
). Simple bath immersion, which is effective for delivery of some traditional inactivated or attenuated vaccines (Corbeil et al., 2000
), has not been found to be effective for DNA vaccines. We are interested in testing the possibility of using transducing baculoviruses to deliver DNA modules to fish by immersion or ingestion, which would be preferred routes of administration in hatchery situations where large numbers of fry need to be vaccinated.
Many of the features of baculoviruses that make them attractive as gene therapy vehicles could potentially be exploited for the development of fish vaccines that could be delivered by immersion or ingestion. The results presented here demonstrate that baculovirus-vectored foreign genes can be expressed in a variety of fish cell lines, providing incentive for further investigations on the development of baculovirus-based vaccines for use in fish.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 September 2002;
accepted 6 January 2003.