Department of Entomology, Clemson University, Clemson, South Carolina 29634, USA1
Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 5M7, Canada2
Author for correspondence: Gerald Carner. Fax +1 864 656 5065. e-mail gcarner{at}clemson.edu
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Abstract |
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Introduction |
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The first ascoviruses were isolated in 1977 from larvae of two different noctuid species, the cotton bollworm, Heliothis zea (Adams et al., 1979 ; Hudson & Carner, 1981
; Carner & Hudson, 1983
) and the clover cutworm, Scotogramma trifolii (Federici, 1982
). Since then, ascoviruses have been isolated from larvae of Autographa precationis (Hamm et al., 1986
), Heliothis virescens (Hamm et al., 1986
), Spodoptera frugiperda (Hamm et al., 1986
) and Trichoplusia ni (Browning et al., 1982
). All these isolates were found within the continental United States. Recently, an ascovirus was isolated from larvae of the leek-moth, Acrolepiopsis assectella, in France (Bigot et al., 1997b
). Since the reported ascoviruses have a relatively wide host-range among noctuid species, ascoviruses are probably distributed worldwide (Federici et al., 1991
). A plausible reason for not having isolated ascoviruses until recently may lie in the pathology of the infected insect. The virus does not cause gross pathology and, therefore, infected insects are not easily identified in the field (Browning et al., 1982
; Hamm et al., 1986
; Federici et al., 1990
).
In this paper, we report the characterization of an ascovirus isolate from Spodoptera exigua in Indonesia, which represents the first isolate from Asia. Its relatedness to the previously reported S. frugiperda ascovirus 1a (SfAV-1a) is described.
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Methods |
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The other ascoviruses used in these studies were SfAV-1a from S. frugiperda (a gift from. B. A. Federici, University of California, Riverside, CA, USA) and TnAV-2 isolated from H. virescens. SfAV-1a was in the form of concentrated vesicles. TnAV-2 is actually a variant of TnAV-2a (Federici et al., 1990 ). TnAV-2 was isolated from H. virescens from a cotton field in South Carolina. It was propagated in H. virescens as described above.
Virus purification and DNA analysis.
Virus-containing vesicles from infected larvae were disrupted by ultrasonication and the virions were purified through sucrose density gradients (Federici et al., 1990 ). Intact viral DNA was purified on CsClethidium bromide gradients by ultracentrifugation.
Viral DNA was digested with restriction endonucleases (REN) (HindIII, EcoRI and PstI) under the conditions recommended by the manufacturers (BioLabs or Promega) and analysed by 0·7% agarose gel electrophoresis with -HindIII DNA and 1 kb DNA ladder (GibcoBRL) as molecular size markers. The gel was stained with ethidium bromide and photographed with an IS-1000 Digital Imaging System (Alpha Innotech Corp., San Leandro, CA, USA). The accompanying software (version 2.02) was used to estimate the molecular mass of DNA fragments
DNA hybridization analyses.
Genomic DNAs (500, 50 and 5 ng) from TnAV-2, SfAV-1a and SeAV-5a were digested with HindIII and EcoRI (Promega or BioLabs) and separated on 0·7% agarose gels. A 1 kb DNA ladder marker was used as a size marker and as a negative control in the following hybridization analyses. Fragments were blotted onto a Hybond N+ nylon membrane (Amersham) (Southern, 1975 ; Chomczynski, 1992
). Genomic DNA probes (40 ng) of TnAV-2, SfAV-1a and SeAV-5a were digested with Sau3AI and the digested products (about 200 bp in length) were labelled with [32P]dATP with a random primer kit (GibcoBRL). Hybridization was carried out in 6x SSC, 0·5% SDS and 5x Denhardts solution at 65 °C for 21 h. The membrane was washed twice in 2x SSC for 5 min at room temperature (23 °C) and twice in 2x SSC0·1% SDS for 30 min at 65 °C.
PAGE and immunological analyses.
Purified virions of each ascovirus were mixed with equal volumes of 2x SDS sample buffer (containing the reducing agent 2-mercaptoethanol), boiled for 2 min and proteins separated on a 12% SDSPAGE discontinuous gel. AcMNPV occlusion bodies were used as a protein size reference and a negative control in immunoblot analysis. Duplicate conditions of the above SDSPAGE without staining were used for Western blot analysis. Separated proteins were transferred onto Nytran nitrocellulose membrane following the standard protocol for Western analysis. A 50 kDa (P50) protein common to all ascoviruses (Federici et al., 1990 ) was cut out of the gel for antibody production. Antiserum was raised in rabbits by Cocalico Biologicals Inc. (Reamstown, PA, USA) and pre-immune serum was tested as a negative control. The P50 antiserum at a 1:5000 dilution was used to bind P50 on the membrane. Bonded antiserum was detected using a Promega ProtoBlot Western blot AP systems kit.
Infectivity and host range of SeAV-5a.
The noctuid larvae tested for determining the host range of SeAV-5a were P. includens, T. ni, H. virescens, S. frugiperda and S. exigua. For each species, 22 to 60 third-instar larvae were injected with an insect pin contaminated with purified SeAV-5a as described above. For controls, pins were dipped in sterile water. Treated larvae were incubated on pinto bean-based artificial diet at 27 °C and 70% relative humidity. Infection was ascertained by observing characteristic vesicles in the haemolymph as described above. Mortality was recorded daily until the treated larvae had either died or pupated. Effects of SeAV-5a infection on S. exigua and S. frugiperda were monitored by weighing infected larvae and control larvae individually until they pupated or died.
Replication of SeAV-5a in Sf-21 cells.
The Sf-21 cell line was kindly provided by A. H. Wood (Boyce Thompson Institute, Cornell University, NY, USA). Cells were maintained as a monolayer in TNM-FH medium in 25 cm2 T-flasks at 27 °C. Haemolymph was collected from the S. exigua larvae at 4 days p.i. by piercing a proleg. Cloudy white haemolymph was collected with a Pasteur pipette, suspended in 0·5 ml of TNM-FH medium and filtered through a 0·45 µm filter. The filtrate was then inoculated into Sf-21 cells at 30% confluence. Cells were monitored daily and infection was confirmed by transmission electron microscopy (TEM).
Electron microscopy.
For negative-stained TEM preparations, purified virions were stained with 2% phosphotungstic acid on a coated grid. Ultrastructure and histopathology were investigated as follows. Third-instar S. exigua were inoculated with SeAV-5a following the method described earlier. At 4 days p.i., haemolymph, fat body and tracheal epidermis were isolated and processed according to Granados & Lawler (1981) . For examination of free vesicles, the haemolymph of infected larvae was harvested and centrifuged as described by Federici et al. (1990)
. The pellet was embedded and processed for electron microscopy.
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Results |
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The response of lepidopteran larvae to infection by SeAV-5a was varied. In the case of S. exigua, larvae were unable to develop into healthy adults and most died in the larval stage. Approximately 71·7% of the P. includens larvae grew to the prepupal stage and a portion (46·7%) of them died in the pupation cocoon. The rest (25%) developed into pupae that were smaller than the healthy ones. Adults that emerged from these smaller pupae showed deformed wings that would not spread out. Most infected S. frugiperda larvae (86·2%) died in the larval stage. Some larvae (13·8%) survived to the pupal stage, but emergence did not occur (Table 1).
The cell line Sf-21 was found to be permissive to SeAV-5a and infection was manifested by early deformation of the cell contour at 2 days p.i. Bulbous protrusions from infected cells formed at 3 days p.i., and the cells eventually lysed and released refractive vesicles (Fig. 5A). We observed that infection was best when inoculation was made at 30% cell confluence. If cells were inoculated at above 60 % confluence, they became overgrown, making it difficult to see infection. Progeny virus (100 µl) harvested from the medium at 5 days p.i. was infectious to healthy Sf-21 cells.
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Ultrathin sections of the tissues of S. exigua infected by SeAV-5a revealed that this ascovirus replicated primarily in fat body cells, not in other tissues examined. Initial replication of SeAV-5a in the nuclei was characterized by the formation of a virogenic stroma and a concomitant deformation of the nuclear and cell membranes. The cell membrane also assumed an irregular shape as the cell became deformed. Some mature virions were observed in the cytoplasm of the infected fat body cells (Fig. 5C). Normally, the virions were contained in vesicles (Fig. 5D
), but sometimes virions were observed within vacuoles in the vesicles (Fig. 5E
). The number of virions in the vacuoles varied from one to five and some vesicles contained empty vacuoles (Fig. 5F
, G
). When fat body tissue was examined over time, no relationship could be established between the number of vacuoles and time p.i. In the process of virion purification, the haemolymph was sonicated and centrifuged. The pellet was found to be composed entirely of virions within vacuoles. When these vacuoles were analysed by SDSPAGE, they exhibited the same peptide pattern as purified virions (data not shown).
SeAV-5a vesicles from S. exigua measured about 5x4 µm, were generally oval shaped and were packed with virions (Fig. 5D, F
). The virions formed clusters in the vesicles and occasionally a big vacuole not containing virions could be seen (Fig. 5F
, G
). Mitochondria were also enclosed in the vesicles and located at the periphery (Fig. 5D
). Vesicles in the infected fat body tissues had rough surfaces while the free vesicles in the haemolymph had a relatively smooth appearance (Fig. 5D
, F
). Aberrant long secondary viral products (about 1·6 µm) were observed in the vesicles (Fig. 5G
) and in some cases the virions were embedded in occlusion bodies (Fig. 5G
, H
). The size of the occlusion body was about 1x1·5 µm.
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Discussion |
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These four accepted ascovirus species could not be cross-hybridized at either high or low stringency conditions. SfAV-1a and HvAV-3a replicate primarily in the fat body tissue of susceptible hosts. TnAV-2a has a wide tissue tropism, replicating in several tissues including the fat body, tracheal matrix and epidermis of infected larvae. DpAV-4a replicates primarily in the pupal stage, where fat body and midgut are the primary tissues attacked (Bigot et al., 1997b ). TnAV-2a and HvAV-3a have a wide experimental host-range among larvae of the Noctuidae. However, the SfAV-1a host-range is restricted primarily to species of Spodoptera, and DpAV-4a infects species of the family Yponomeutidae. SfAV-1a virions are bacilliform and can be found occluded in vesiculate occlusion bodies, whereas TnAV-2a virions are allantoid and are not occluded in occlusion bodies. HvAV-3a virions are ovoidal to bacilliform, and are not occluded in occlusion bodies (Federici et al., 1999
).
SeAV-5a could not hybridize to TnAV-2, but some regions of the genome hybridized to SfAV-1a (Fig. 2B). Hybridization of SeAV-5a to HvAV-3a and DpAV-4a was not performed because of the unavailability of the viruses. It is unlikely that this new ascovirus from S. exigua is the same as DpAV-4a because it has a much larger genome. Also, host range and pathology are quite different between SeAV-5a and DpAV-4a (Bigot et al., 1997a
, b
). In addition to the partial hybridization to SfAV-1a, SeAV-5a formed an occlusion body structure similar to that of SfAV-1a but with a different matrix structure (Fig. 5D
, G
, H
). The occlusion body of SfAV-1a has a vesiculate structure, but the occlusion body of SeAV-5a resembles that of a baculovirus polyhedron, which lacks the vesiculate structure (Federici et al., 1990
; Fig. 5G
, H
). SeAV-5a has a wider host range than SfAV-1a, which is restricted to the genus Spodoptera (Hamm et al., 1986
; Table 1
). The wide host range of SeAV-5a resembles that of HvAV-3a, but the former does not infect H. virescens larvae and the latter does not form occlusion bodies (Table 1
). Thus, the ascovirus isolate from S. exigua could not be assigned to any of the four reported ascoviruses in the present classification scheme and therefore has been tentatively assigned a new species code (SeAV-5a) in this report.
Since Southern and Western analysis (Fig. 2B, C
, Fig. 3B
) showed some relatedness between SeAV-5a and SfAV-1a, SeAV-5a may be related more closely to SfAV-1a than to TnAV-2. However, previous reports of dot blot and Southern hybridization did not establish this relatedness between SfAV-1a and other ascoviruses (Federici et al., 1990
; Hamm et al., 1998
). It is still not known how closely SeAV-5a and SfAV-1a are related, since genome organization and gene sequences are not well characterized. At present, only the DNA polymerase gene sequence of SfAV-1a has been published (Pellock et al., 1996
). In Southern blot analysis, particular REN fragments of SfAV-1a hybridized to SeAV-5a DNA and vice versa (Fig. 2B
, C
). This suggests that certain region(s) or gene(s) may be conserved between the two viruses. It is conceivable that the genes encoding P50 hybridized with each other since the homologous proteins have conserved antigenic sites (Fig. 3B
). Unless the two hybridizing fragments in each digest are contiguous on the genome, it appears that there are other regions of high homology between SfAV-1a and SeAV-5a (Fig. 2B
, C
).
It is curious that some SeAV-5a virions were embedded in the occlusion body structure within the vesicle (Fig. 5D, G
, H
). Some were also found embedded in a similar structure at the periphery of the vesicles (Fig. 5D
). The function of these occlusion bodies may be similar to that of baculoviruses, namely to protect the virus from adverse conditions outside the host (Federici et al., 1990
).
A novel structure consisting of vacuoles enclosing several virions was observed in the vesicles (Fig. 5E). This structure was resistant to the ultrasonication employed in breaking the vesicles to release virions. SDSPAGE analysis of the protein composition of this structure did not show any additional proteins compared with those found in virions, which may indicate that they do not contain protein (data not shown). The vacuoles may be formed by invagination of plasma membrane of the infected cells. The function of this structure is unknown but it may protect the virions in the environment.
The effects of ascoviruses on the development and growth of infected hosts have been documented for other isolates (Govindarajan & Federici, 1990 ; Carner & Hudson, 1983
; Hamm et al., 1986
; Federici & Govindarajan, 1990
). SeAV-5a infection arrested development in all susceptible hosts (Fig. 4
, Table 1
). The response of the various insect species to SeAV-5a was reflected by changes in the appearance of the haemolymph. In all susceptible species (S. exigua, S. frugiperda, T. ni and P. includens) the haemolymph turned milky white. In the future, it may be useful to correlate haemolymph whiteness with level of virus production. The mechanism of larval growth retardation by ascoviruses has not been elucidated, but bears marked resemblance to that caused by polydnaviruses. The latter group of viruses appears to reduce synthesis of ecdysone in the prothoracic gland (PTG), and consequently extends the larval life span (Dover et al., 1988
). Whether ascoviruses have the same effect on the PTG remains to be shown.
Infection of SeAV-5a in vitro to cell line Sf-21 should provide a vehicle to study this group at the cellular level. The effect of SeAV-5a on Sf-21 is similar to that of SfAV-1a (Federici & Vlak, 1986 ). Techniques for transfection of cells with viral DNA need to be developed to allow production of recombinant viruses for gene expression studies.
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Acknowledgments |
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Footnotes |
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References |
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Received 26 May 2000;
accepted 14 August 2000.
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