Department of Plant and Microbial Biology, 251 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA
Correspondence
Loy E. Volkman
lvolkman{at}nature.berkeley.edu
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ABSTRACT |
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Present address: Department of Cell and Tissue Biology, 521 Parnassus, Room C741, University of California, San Francisco, CA 94143-0422, USA.
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INTRODUCTION |
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Whilst AcMNPV has a taxonomically broad host range, susceptibility varies significantly among species. Some species, such as S. frugiperda, are so resistant to oral infection that they cannot be controlled effectively in the field, even though they exhibit no systemic resistance to AcMNPV when budded virus is introduced intrahaemocoelically (IH) (Haas-Stapleton et al., 2003). Pathogenesis studies using an AcMNPV recombinant carrying the lacZ reporter gene have shown that the inability of AcMNPV ODV to infect midgut cells is a significant barrier to oral infection of S. frugiperda (Haas-Stapleton et al., 2003
). We therefore postulated that this midgut barrier may be due to the lack of specific AcMNPV ODV binding and/or fusion with S. frugiperda midgut columnar cells. To test this hypothesis, we used an in vivo fluorescence-dequenching assay (Haas-Stapleton et al., 2004
) to compare the ODV binding and fusion properties of AcMNPV with those of the homologous virus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). SfMNPV has a very narrow host range that includes only three Spodoptera species, all of which are highly susceptible to mortal infection (Adams & McClintock, 1991
; Hamm & Styer, 1985
).
In bioassays, we found that all of the SfMNPV isolates that were provided to us by colleagues exhibited extremely low oral infectivity; therefore, we cloned and characterized a highly virulent strain of SfMNPV (SfMNPV-UC2) from an S. frugiperda cadaver collected in Louisiana, USA. Compared to the ODV of SfMNPV-UC2, AcMNPV ODV binding to the midgut of S. frugiperda was both quantitatively and qualitatively different, which could account for the vastly different oral infectivities.
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METHODS |
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SfMNPV cloning and purification.
Because the SfMNPV isolates provided to us by other laboratories lacked oral infectivity in S. frugiperda larvae, Dr James Fuxa kindly provided us with baculovirus-killed S. frugiperda cadavers that were field-collected in Louisiana, USA. To maintain oral infectivity, an SfMNPV isolate was cloned from these cadavers by using in vivo techniques. Briefly, occlusions from the field-collected cadavers were partially purified by sucrose-density centrifugation (Summers & Smith, 1987), suspended in sterile ddH2O and then diluted serially in a neutrally buoyant solution of sterile glycerin and water (3 : 2, v/v) (Washburn et al., 1995
). Cohorts of 32 5° S. frugiperda larvae were inoculated orally with 1 µl of each dilution and from the cohort that generated <10 % mortality, we selected one virus-killed cadaver for the second round of cloning. Occlusions from this cadaver were diluted serially, the bioassay scheme was repeated, and a single cadaver was again selected from a cohort with <LD10. This cadaver was used to lace diet that was then fed to 5° S. frugiperda larvae. Five days later, individual larvae were bled, and their haemocytes were examined for viral occlusions by using light microscopy (400x). We removed the haemolymph from one infected larva, pelleted the haemocytes by centrifugation and collected the supernatant containing the BV by following the methods of Trudeau et al. (2001)
. The BV stock was then diluted serially in Grace's medium containing 1 % fetal bovine serum (FBS) and 0·1 % 1-phenyl-2-thiourea. Cohorts of 32 feeding, fifth-instar S. frugiperda were IH-injected with each serial dilution and a single cadaver from the dilution that generated <10 % mortality was again used to lace diet. This IH-cloning process was repeated two additional times, resulting in a putative isolate of SfMNPV (designated SfMNPV-UC2).
In order to determine whether SfMNPV-UC2 was actually a cloned isolate of SfMNPV, genomic DNA was isolated from gradient-purified ODV and subjected to cleavage by four different restriction enzymes (EcoRI, HindIII, PstI and SacI) by using standard molecular biological techniques (Sambrook & Russell, 2001). The cleaved fragments were electrophoresed in a 0·7 % agarose gel and the DNA-cleavage patterns were compared to those of DNA isolated from a known strain of SfMNPV (SfMNPV-LA) and AcMNPV-hsp70/lacZ. For further confirmation that our isolate was SfMNPV, we also conducted limited host-range studies.
Viruses and virus preparation.
SfMNPV-UC2, SfMNPV-LA (provided by Bruce Hammock, University of California, Davis, CA, USA) and the AcMNPV recombinant virus AcMNPV-hsp70/lacZ were used in this study. AcMNPV-hsp70/lacZ contains all of the genes found in the wild-type virus, plus the -galactosidase reporter gene driven by the Drosophila hsp70 promoter. This AcMNPV recombinant has wild-type virulence in vivo (Engelhard et al., 1994
).
Occlusions of SfMNPV and AcMNPV-hsp70/lacZ were isolated from virus-killed cadavers of S. frugiperda and T. ni, respectively, and partially purified as described above. Occlusion populations were quantified by using a haemocytometer and maintained at 4 °C until use. For some experiments, ODV was isolated by using methods described previously (Volkman et al., 1976) with the following modifications. The ODV pellets were resuspended in minimal volumes of PBS (137 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 1 mM KH2PO4, pH 7·4), the total volumes were measured and samples were removed to determine the protein concentrations by using the BCA protein assay (Pierce). Subsequently, 1 % FBS was added for stabilization and the ODV suspensions were aliquotted into microfuge tubes for storage at 80 °C until use. AcMNPV-hsp70/lacZ BV was harvested from the medium of infected Sf-9 cultured insect cells 3 days post-infection and titres were determined by immunoplaque assay using Sf-9 cells (Volkman & Goldsmith, 1982
). SfMNPV-UC2 BV was generated by orally inoculating 5° S. frugiperda with SfMNPV-UC2 occlusions. After incubation for 6 days at 28 °C, eight larvae were bled into ice-cold Grace's culture medium containing 10 % FBS. BV was harvested and diluted serially as described above and quantified by immunoplaque assay on Sf-9 cells (Volkman & Goldsmith, 1982
).
R18 labelling.
For binding/fusion assays, ODV was labelled with the self-quenching fluorescent probe octadecyl rhodamine B chloride (R18; Molecular Probes) as described by Nussbaum & Loyter (1987), with the following modification. Unbound R18 was eliminated by dialysis using a 10K MWCO dialysis cassette (Pierce) in 1·5 l PBS for 1216 h at 4 °C in the dark (Haas-Stapleton et al., 2004
). Under these conditions, R18 was inserted into the viral envelope at self-quenching levels. We confirmed that all residual unbound R18 was eliminated from the ODVR preparations as described previously (Haas-Stapleton et al., 2004
). For each ODV preparation, a sample was removed to determine fluorescence (µg ODVR protein)1, i.e. specific fluorescence (Haas-Stapleton et al., 2004
). Among preparations, this value ranged between 1·6x106 and 3·8x106 fluorescence units (µg ODVR protein)1, but for all experiments with paired viruses (e.g. AcMNPV-hsp70lacZ vs SfMNPV-UC2), the specific fluorescence values of both ODVs were equivalent (data not shown). All ODVR stocks were diluted to
1·5 mg ml1 in PBS with 1 % FBS and stored at 4 °C until use. Mock-labelled ODV was prepared similarly, but in the absence of R18 probe.
Quantification of ODVR binding and fusion to the midgut.
For binding and fusion assays, 4° or 5° larvae were inoculated orally with 2 µl ODVR or PBS and incubated at 28 °C in the dark without food. After 1 h, the midgut from each larva was removed and the midgut epithelium was separated from the basal lamina and stored at 80 °C in separation buffer as described previously (Haas-Stapleton et al., 2004). Subsequently, the amount of ODVR bound and fused was quantified by using a Fluorolog-3 fluorescence spectrophotometer (Instruments S.A.) (EX 560/EM 583) (Haas-Stapleton et al., 2004
). To compare binding and fusion of AcMNPV-hsp70/lacZ and SfMNPV-UC2 ODV, 3 µg ODVR was inoculated orally into 5° S. frugiperda and 4° H. virescens (n=2147 larvae per treatment). For competition experiments, 5° S. frugiperda larvae were inoculated orally with 2 µg SfMNPV-UC2 ODVR, with or without unlabelled ODV competitor (i.e. AcMNPV-hsp70/lacZ or SfMNPV-UC2). All results were analysed by ANOVA and a Fisher's protected least significant difference test was used to discriminate significant differences among means (STATVIEW version 5.0.1).
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RESULTS |
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In vivo cloning of SfMNPV-UC2
To determine whether SfMNPV-UC2 was a new isolate of SfMNPV and also to evaluate its purity, genomic DNA was extracted from gradient-purified ODVs of SfMNPV-UC2, SfMNPV-LA and AcMNPV-hsp70/lacZ. The extracted DNAs were digested with the restriction enzymes EcoRI, HindIII, PstI and SacI and subjected to agarose-gel electrophoresis. The digest patterns from SfMNPV-UC2 and SfMNPV-LA were very similar to each other (and to published profiles of SfMNPV DNA; Maruniak et al., 1984), but distinct from those of AcMNPV-hsp70/lacZ (Fig. 1
). The most striking differences among the SfMNPV strains were revealed in the SacI digest, in which the SfMNPV-LA strain produced an extra band of
7 kbp (arrow, Fig. 1
). In addition, a submolar band of
7·5 kbp (barely visible) was evident in the SacI digests of SfMNPV-LA, suggesting that this isolate was not pure. In contrast, no submolar bands were detected in digests of the UC2 strain, indicating that this strain was pure (Fig. 1
). The similarity in the digest pattern of SfMNPV-UC2 to that of the other SfMNPVs provided strong empirical evidence that the new isolate was a strain of SfMNPV.
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DISCUSSION |
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Results from a previous investigation of AcMNPV pathogenesis in fifth-instar S. frugiperda larvae demonstrated that, whilst BV is highly infectious in the haemocoel, ODV infects midgut cells with extremely low efficiency (Haas-Stapleton et al., 2003). To define the nature of the midgut barrier to oral infection by AcMNPV, we compared the ODVs of SfMNPV-UC2 and AcMNPV-hsp70/lacZ in the initial steps of primary infection (binding and fusion with target midgut cells) by using a fluorescence-dequenching assay. Our results indicated that SfMNPV ODV bound to a midgut cell receptor(s) that was not recognized by AcMNPV ODV and that this specificity of binding of SfMNPV ODV with S. frugiperda midgut cells may play an important role in oral infection. It is interesting to note that, once the ODVs of SfMNPV and AcMNPV had bound to midgut cells, the fusion efficiencies were similar. This result indicated that the specificity of viral binding was not a requirement for fusion, and that fusion, per se, was not sufficient to insure productive infection.
The virulence phenotype of having very low oral ODV infectivity and high intrahaemocoelic BV infectivity describes both wild-type AcMNPV in S. frugiperda and a p74-deficient mutant of AcMNPV in larvae of both T. ni and H. virescens (Faulkner et al., 1997; Haas-Stapleton et al., 2004
). When the binding and fusion properties of the p74 mutant and wild-type ODVs to midgut epithelial cells in H. virescens were compared, the mutant bound at one-third of the level of the wild-type. However, in that system, as in this one, there was also a significant qualitative difference in binding. The ODV of the p74 mutant could not compete with parental ODV for midgut receptor-binding sites (Haas-Stapleton et al., 2004
). Together, the results of both of these studies suggest that specificity of binding can play an important role in ODV infection of midgut cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 December 2004;
accepted 1 February 2005.
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