Department of Animal Health and Biomedical Sciences, University of WisconsinMadison, 1656 Linden Drive, Madison, WI 53706, USA
Correspondence
Bruce Christensen
christensen{at}svm.vetmed.wisc.edu
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ABSTRACT |
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INTRODUCTION |
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The only member of the Nodaviridae family shown to infect mosquitoes is nodamura virus (NOV; Ball & Johnson, 1998; Hendry, 1991
). NOV infects cell lines obtained from two mosquito species, Aedes albopictus and Ae. aegypti. No CPE is observed but infectious virus is produced at a low level (Bailey et al., 1975
). No data are available on virus titres in these species because a plaque assay is not available for NOV. However, NOV is lethal to honey bees (Bailey & Scott, 1973
) and suckling mice (Scherer & Hurlbut, 1967
) and, unlike FHV, is considered pathogenic to animals.
FHV has the smallest genome of any positive-strand animal RNA virus and has been studied extensively at the molecular level. The genome of FHV consists of two RNAs packaged in a single, non-enveloped, icosahedral virion. RNA1 (3·1 kb) encodes the viral polymerase (protein A; 112 kDa) and RNA2 (1·4 kb) encodes the viral capsid protein precursor (43 kDa). RNA1 is capable of independent replication but the replication of RNA2 is RNA1-dependent (Gallagher et al., 1983). Formation of progeny virus particles requires co-infection of cells with both RNAs.
At its 3' end, FHV RNA1 encodes a subgenomic RNA3 (0·4 kb) containing two overlapping open reading frames (ORFs) encoding proteins B1 and B2. The translation of B1 is inefficient and B1 is not required for the FHV life cycle. Protein B2, translated from the second AUG in RNA3, accumulates at a high level (5 % of total cell protein) in Drosophila cells at early stages of infection and is not packaged into the virus particle (Friesen & Rueckert, 1982). Protein B2 is not required for RNA1 replication or RNA3 synthesis (Ball, 1995
); it was recently shown to suppress RNA silencing in Drosophila cells and in transgenic plants (Li et al., 2002
). Autonomous replication of FHV RNA1 and its robust capacity to synthesize viral RNAs and subgenomic RNA3 provides a promising approach towards amplifying heterologous sequences in insect cells expressed from vectors. Price et al. (2000)
developed a yeast DNA plasmid system expressing wild-type FHV RNA1 in vivo and used this system to express green fluorescent protein (GFP) in yeast.
The major protein produced during FHV infection is capsid protein precursor alpha, translated from RNA2. Analysis of deletion mutants has revealed cis-acting sequences required for the replication of FHV RNA2 (Ball & Li, 1993) and has shown that much of the sequence encoding the capsid protein can be deleted without severely affecting subsequent virus replication (Zhong, 1993
). A defective interfering (DI) RNA (DI 634) produced after high multiplicity infection in Drosophila cells is a deletion mutant of FHV RNA2 containing 634 bases out of 1400 bases of RNA2. DI 634 accumulates to higher levels in infected tissues than genomic RNAs (R. Dasgupta & R. R. Rueckert, unpublished observation). Vectors derived from the cDNA clones of both FHV RNA2 and DI 634 have been used to express reporter genes such as CAT and GFP in Drosophila cells and yeast (Zhong et al., 1992
; Price et al., 2000
, 2002
).
We wanted to explore the possibility of developing FHV-based vectors for transient gene expression and gene silencing in mosquitoes. Stable transformation of insects based on transposable elements, such as Mariner, Hermes and piggybac, remains a laborious procedure (see review by Atkinson et al., 2001). Transient expression systems, on the other hand, are a faster and more efficient means of studying gene expression as well as gene silencing. Sindbis (SIN) virus vectors have been the most widely used viruses for gene expression in mosquitoes (Higgs et al., 1999
; Olson et al., 1998
, 2000
). However, certain limitations exist for the SIN virus system: (i) SIN virus does not disseminate and replicate in all mosquito tissues; (ii) recombinant SIN virus has a size limitation of about 1 kb for inserting a gene of interest; and (iii) it is a human pathogen. Therefore, the development of other viral expression systems that are non-pathogenic to humans and preferably with a broader insect host range will be beneficial and will increase the repertoire of transducing virus expression systems. FHV-based vectors can provide both of these advantages, and the bipartite genome of FHV, which carries out distinct functions, could be engineered to express two different genes simultaneously.
Here we report that FHV replicates in a mosquito cell culture as well as in four species of mosquito. The mosquitoes tested for FHV growth are vectors of parasites (i.e. Anopheles gambiae, Culex pipiens pipiens) and viruses (i.e. Ae. aegypti, C. pipiens pipiens) that have a significant impact on global public health, or are model organisms for studies of mosquito immune responses (i.e. Armigeres subalbatus). We have also demonstrated that a vector derived from a cDNA clone of FHV DI RNA is capable of expressing GFP in mosquito cells and tissues.
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METHODS |
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Cells, propagation of virus and extraction of virion RNA.
D. melanogaster cells (DL1) were grown at 26 °C in Schneider's insect cell medium (Schneider, 1972) supplemented with 10 % foetal bovine serum (FBS). C6/36 cells (derived from Ae. albopictus; ATCC CCL-126) were maintained in Leibovitz L-15 medium containing 10 % FBS and were grown at 28 °C in close-capped tissue culture flasks. FHV was propagated in DL1 cells, purified by sucrose gradient centrifugation and virus titre was determined by plaque assay on Drosophila cell monolayers (Selling & Rueckert, 1984
). DL1 cells infected with FHV at an m.o.i. of 0·11 for 48 h were the source of virus for blood feeding and injecting mosquitoes. FHV RNA isolated from purified virus by phenol extraction was used for liposome-mediated transfection of insect cells.
Growth of FHV in C6/36 mosquito cells.
C6/36 cells were passaged the day before inoculation and allowed to reach 8590 % confluency before inoculation with FHV at an m.o.i. of 1 (inocula were approximately 1·7x106 p.f.u.). FHV was obtained from the supernatant of virus-infected Drosophila cells. After 1 h incubation at 28 °C, the inoculum was removed and cells were washed twice with fresh medium, then incubated at 28 °C for up to 96 h. Cell supernatant was collected at 24 h intervals post-infection after two rounds of freeze-thawing to release intracellular viruses and centrifugation to remove cell debris. Virus titre was determined by plaque assay on Drosophila cells. Inoculated C6/36 cells were also subjected to an immunofluorescence assay (IFA).
In order to clone the subsets of C6/36 cells that support optimum growth of FHV, C6/36 cells were serially diluted and seeded on a six-well plate to obtain a single-cell suspension; cells were then overlaid with medium containing agar. Individual colonies were isolated after 710 days, transferred to microtitre wells and allowed to grow for an additional 3 days. Cells from individual clones were inoculated with FHV at an m.o.i. of 1, fixed on glass slides after 72 h and FHV growth was monitored by IFA. Cells that promoted enhanced growth were further subcloned to obtain a clonal cell line that supported maximum growth of FHV.
Infection of mosquitoes with FHV.
Supernatants from FHV-infected Drosophila cells were used to infect mosquitoes by injection (Beernsten & Christensen, 1990). Individual mosquitoes were injected with 0·51 µl containing approximately 5x1045x105 p.f.u. infectious virus. The same source of FHV was used to infect mosquitoes per os. Mosquitoes were exposed to FHV mixed with an artificial blood meal (Kogan, 1990
) at a 1 : 1 ratio (virus titres were 12x108 p.f.u. ml-1) through a Parafilm membrane or chicken skin on a water-jacketed membrane feeder (Rutledge et al., 1964
).
Detection of FHV growth.
FHV growth was monitored in both cell cultures and mosquitoes using an IFA and a plaque assay on Drosophila cells. Control and virus-exposed mosquito tissues were removed by dissection and assayed for the growth of virus by IFA (Cheng et al., 2001). Polyclonal anti-FHV antibody (raised against capsid protein) and FITC-labelled goat anti-rabbit IgG at dilutions of 1 : 400 and 1 : 800, respectively, were used as primary and secondary antibodies for virus detection. The fluorescence was examined in cells or tissues with UV illumination using an Olympus IX70 inverted microscope. For plaque assays, five virus-exposed mosquitoes from each species were homogenized with a plastic pestle in 1·5 ml Eppendorf tubes in isotonic buffer (IB; 100 mM NaCl, 35 mM Pipes, 10 mM KCl, 1 mM MgCl2, 1 mM CaCl2, pH 6·8). Supernatant was collected after centrifugation and plaque assays were performed after di;uting 104105 in IB. The resultant titre was divided by five to determine approximate p.f.u. per mosquito.
Plasmid constructs for expression of GFP.
cDNA clones of FHV DI RNA in pBluescript (pDI634; Zhong et al., 1992; Zhong, 1993
) and pEGFP (Clonetech) were used to construct pDIeGFP (Fig. 1
C). The eGFP ORF (nt 2891105) was amplified from pEGFP by PCR using the eGFP-specific primers 5'-GTCGAGCTCAAATGGTGAGCAAGGGCG-3' and 5'-GTCGAGCTCTCTTGTACAGCTCGTCC-3', both containing SacI sites at their 5' ends. The PCR product was then transferred to the SacI site at nt 58 of FHV RNA2 (Dasgupta & Sgro, 1989
; GenBank accession no. X15959) using standard techniques (Sambrook et al., 1989
). FHV DI RNA contained nt 1249, 517728 and 12281400 of RNA2 (Fig. 1A, B
) and contained all the signals for replication and encapsidation (Zhong et al., 1992
).
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Drosophila or C6/36 cells, at 5070 % confluency in a six-well plate, were transfected with 13 µg RNA transcripts using the TransMessenger transfection reagent (Qiagen) according to the manufacturer's protocol. Approximately 0·5 µg purified FHV RNA was used for co-transfection in each experiment. Cells were monitored for GFP expression up to 96 h post-transfection. Supernatant and cells were subjected to two cycles of freeze-thawing to release virus particles from transfected cells. Green fluorescence was monitored in cells after 4872 h by UV illumination using an Olympus IX70 inverted microscope and images were captured by a Spot RT slider digital camera (Diagnostic Instruments, Inc.).
In vivo expression of GFP in mosquitoes.
FHVDIeGFP virus was generated from Drosophila cells co-transfected in vitro with pDIeGFP transcripts and FHV genomic RNAs. Virus was harvested from cells at 72 h post-transfection after two cycles of freezethawing and subjected to centrifugation at 8000 r.p.m. for 10 min to remove cell debris. The supernatant was inoculated into Ae. aegypti either directly or after concentration by ultracentrifugation. To concentrate the FHVDIeGFP virus, supernatant from transfected cells was first pelleted over a 30 % sucrose solution and resuspended in 4 ml STE buffer (10 mM Tris/HCl, 10 mM NaCl, 1 mM EDTA, pH 7·5). Virus was pelleted again by ultracentrifugation and resuspended in 40 µl STE resulting in an increase in virus concentration of 100-fold. Approximately 0·5 µl of the resuspended virus was injected into individual Ae. aegypti mosquitoes. Mosquitoes were monitored at 3, 7, 10 and 14 days post-injection and various tissues were dissected and examined for GFP expression.
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RESULTS AND DISCUSSION |
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Three rounds of subcloning produced a C6/36 clone #8-12-8 that showed a dramatic increase (approximately 100-fold) in the ability to amplify FHV compared with the original cells, as measured by virus titration (Table 1). IFA (Fig. 2
) with anti-FHV antibodies also confirmed that more C6/36 cells in the cloned cell line #8-12-8 were infected. The growth of original C6/36 cells versus the cloned cells showed no considerable differences: the cell doubling time was roughly the same (2436 h) and the fluorescence intensity of individual infected cells was similar between the original and cloned cell line. Collectively, these data indicated that FHV replicated equally well in infected cells from both cell lines but that the cloned cell line had more cells susceptible to FHV infection. No CPE was detected in the FHV-infected C6/36 cells. This clonal cell population, selected for susceptibility to FHV infection, could be valuable for studies of physiological processes in mosquito cell lines.
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FHV vectors express the reporter gene GFP in vitro in Drosophila cells and mosquito cells
We inserted the sequence of eGFP near the 5' end (nt 58) of the DI RNA so that eGFP was efficiently translated using the ATG (nt 23) and leader sequence of the FHV capsid protein (Fig. 1). The transcript made from linearized pDIeGFP was 1364 bases long and contained an ORF of 1203 bases. The resulting fusion protein (401 aa) contained the first 13 aa of the FHV capsid protein followed by eGFP (239 aa), part of the FHV capsid protein (75 aa coded by nt 24249 of the DI RNA) and a random sequence of 74 aa at the C terminus.
GFP expression was evident in Drosophila cells that were co-transfected with this transcript and FHV virion RNAs. We observed a high level of GFP expression in Drosophila cells, which increased with time and peaked at 72 h post-transfection (Fig. 5A). We reinfected fresh cells with supernatants from these transfected cell lysates and detected FHV and GFP expression at different time points (Fig. 5B
) confirming that virus particles were made in Drosophila cells and GFP sequences were retained in the progeny viruses.
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FHV vectors express the reporter gene GFP in vivo in Ae. aegypti
When supernatant from Drosophila cells transfected with transcripts derived from pDIeGFP was used to infect mosquitoes by injection in an in vivo experiment, GFP expression was detected in five mosquitoes out of 40 examined (data not shown). In contrast, when supernatants were concentrated by ultracentrifugation and injected into mosquitoes, GFP expression was detected in 17 out of 50 mosquitoes tested. Of the 17 mosquitoes, GFP fluorescence was observed in the head tissue of 12, the fat body of nine, the midgut of seven and the salivary glands of five mosquitoes. A representative collection of these images is shown in Fig. 6.
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Our data show that FHV is capable of replicating both in cultured mosquito cells and in vivo in mosquito tissues including the midgut, head tissue, fat body and salivary glands without signs of pathogenicity. In addition, the replication machinery of FHV can be used to express foreign sequences in mosquitoes. Our findings extend the host range of FHV to another order of insects (Diptera) and raise the possibility that FHV will replicate in various insects. Preliminary studies have demonstrated that FHV multiplies to high titres in tsetse flies (Glossina moristans) and expresses viral antigen in various tissues (data not shown). These data, in addition to the small size and wide host range of FHV, suggest that FHV-based vectors could prove extremely useful for studying insect molecular biology, gene expression (with the possibility of expressing two genes simultaneously) and gene silencing.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bailey, L. & Scott, H. A. (1973). The pathogenicity of nodamura virus for insects. Nature 241, 545.[Medline]
Bailey, L., Newman, J. F. E. & Porterfield, J. S. (1975). The multiplication of nodamura virus in insect and mammalian cell culture. J Gen Virol 26, 1520.[Abstract]
Ball, L. A. (1995). Requirements for the self directed replication of flock house virus RNA1. J Virol 69, 720727.[Abstract]
Ball, L. A. & Li, Y. (1993). Cis-acting requirements for the replication of flock house virus RNA 2. J Virol 67, 35443551.[Abstract]
Ball, A. & Johnson, K. L. (1998). Nodaviruses of insects. In The Insect Viruses, pp. 225267. Edited by L. K. Miller & L. A. Ball. New York: Plenum Publishing.
Ball, L. A., Hendry, D. A., Johnson, J. E., Rueckert, R. R. & Scotti, P. D. (2000). Family Nodaviridae. In Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses, pp. 747755. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Beernsten, B. T. & Christensen, B. M. (1990). Dirofilaria immitis: effect on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against microfilariae. Exp Parasitol 71, 406414.[Medline]
Beernsten, B. T., James, A. A. & Christensen, B. M. (2000). Genetics of mosquito vector competence. Microbiol Mol Biol Rev 64, 115137.
Cheng, L. L., Bartholomay, L. C., Olson, K. E., Lowenberger, C., Vizioli, J., Higgs, S., Beaty, B. J. & Christensen, B. M. (2001). Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus. J Insect Sci 1(10), 8.[Medline]
Christensen, B. M. & Sutherland, D. R. (1984). Brugia pahangi: exsheathment and midgut penetration in Aedes aegypti. Trans Am Microsc Soc 103, 423433.
Dasgupta, R. & Sgro, J.-Y. (1989). Nucleotide sequences of three nodavirus RNA2s: the messengers for their coat protein precursors. Nucleic Acids Res 17, 75257526.[Medline]
Dasgupta, R., Garcia, B. H. & Goodman, R. M. (2001). Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes. Proc Natl Acad Sci U S A 98, 49104915.
Friesen, P. D. & Rueckert, R. R. (1982). Black beetle virus: messenger RNA for protein B is a subgenomic viral RNA. J Virol 42, 986995.
Gallagher, T. M., Friesen, P. D. & Rueckert, R. R. (1983). Autonomous replication and expression of RNA1 from black beetle virus. J Virol 46, 481489.
Gerberg, E. J., Barnard, D. R. & Ward, R. A. (1994). Procedures for laboratory rearing of specific mosquitoes. In Manual for Mosquito Rearing and Experimental Techniques, pp. 2365, Bulletin No. 5. Louisiana: American Mosquito Control Association.
Hendry, D. A. (1991). Nodaviridae of invertebrates, In Viruses of Invertebrates, pp. 227276. Edited by E. Korstak. New York: Marcel Dekker.
Higgs, S., Oray, C. T., Myles, K., Olson, K. E. & Beaty, B. J. (1999). Infecting larval arthropods with a chimeric, double subgenomic Sindbis virus vector to express genes of interest. Biotechniques 27, 908911.[Medline]
Kogan, P. H. (1990). Substitute blood meal for investigating and maintaining Aedes aegypti (Diptera: Culicidae). J Med Entomol 27, 709712.[Medline]
Li, H., Li, W. X. & Ding, S. W. (2002). Induction and suppression of RNA silencing by an animal virus. Science 296, 13191321.
Olson, K., Beaty, B. & Higgs, S. (1998). RNA virus expression vectors. In The Insect Viruses, pp. 371404. Edited by L. K. Miller & L. A. Ball. New York: Plenum Publishing.
Olson, K. E., Myles, K. M., Seabaugh, R. C., Higgs, S., Carlson, J. O. & Beaty, B. J. (2000). Development of a Sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of Aedes aegypti following per os infection. Insect Mol Biol 9, 5765.[CrossRef][Medline]
Price, B. D., Rueckert, R. R. & Ahlquist, P. (1996). Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93, 94659470.
Price, B. D., Roeder, M. & Ahlquist, P. (2000). DNA-directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis. J Virol 74, 1172411733.
Price, B. D., Ahlquist, P. & Ball, L. A. (2002). DNA-directed expression of an animal virus RNA for replication-dependent colony formation in Saccharomyces cerevisiae. J Virol 76, 16101616.
Rutledge, L. C., Ward, R. A. & Gould, D. J. (1964). Studies on feeding response of mosquitoes to nutritive solutions in a new membrane feeder. Mosq News 24, 407419.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scherer, W. F. & Hurlbut, H. S. (1967). Nodamura virus from Japan: a new and unusual arbovirus resistant to diethyl ether and chloroform. Am J Epidemiol 86, 271285.[Medline]
Schneider, I. (1972). Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27, 353365.[Medline]
Scotti, P. D., Dearing, S. & Mossop, D. W. (1983). Flock house virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae). Arch Virol 75, 181189.[Medline]
Selling, B. H. & Rueckert, R. R. (1984). Plaque assay for black beetle virus. J Virol 51, 251253.[Medline]
Selling, B. H., Allison, R. F. & Kaesberg, P. (1990). Genomic RNA of an insect virus directs synthesis of infectious virions in plants. Proc Natl Acad Sci U S A 87, 434438.[Abstract]
Zhong, W. (1993). Flock house virus, a small insect ribovirus: replication and encapsidation of RNA2. PhD thesis, University of WisconsinMadison, USA.
Zhong, W., Dasgupta, R. & Rueckert, R. (1992). Evidence that the packaging signal for nodaviral RNA2 is a bulged stemloop. Proc Natl Acad Sci U S A 89, 1114611150.[Abstract]
Received 31 October 2002;
accepted 6 March 2003.