1 Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA
2 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
3 Department of Molecular Biology, University of Wyoming, Laramie, WY 82071-3944, USA
Correspondence
Jan O. Washburn
janwash{at}nature.berkeley.edu
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ABSTRACT |
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INTRODUCTION |
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While AcMNPV ODV and BV are genetically identical and share subsets of structural polypeptides, some polypeptides are distinct. These structural differences undoubtedly relate to the fact that the two viral phenotypes target different types of cells, infect under very different physiological conditions and use different mechanisms to penetrate their host cells (Braunagel & Summers, 1994; Volkman, 1983
, 1997
). GP64 is the major BV envelope glycoprotein of the Group I NPVs and is acquired when BV particles bud through the plasma membrane (Blissard & Rohrmann, 1989
; Volkman et al., 1984
; Whitford et al., 1989
). GP64 is essential for AcMNPV BV infectivity (Monsma et al., 1996
) and has a highly unusual temporal pattern of expression, being synthesized during both early and late phases of infection. This expression pattern is unlike that of all other AcMNPV structural proteins (and indeed viral structural proteins in general), which are only synthesized during the late phase of infection (Blissard & Wenz, 1992
; Hefferon et al., 1999
; Monsma et al., 1996
; Oomens & Blissard, 1999
; Volkman, 1986
). While GP64 is essential for infection, elimination of its early synthesis impacts neither the timing nor the amount of BV produced in vitro and has no effect on BV virulence in vivo (Washburn et al., 2003a
). Interestingly, while GP64 is not a component of AcMNPV ODV, its early synthesis is a significant virulence factor during oral infection of the permissive host, Heliothis virescens (Washburn et al., 2003a
).
The key to understanding this apparent paradox lies in a second trait shared by AcMNPV and other MNPVs: multiple nucleocapsids within a single ODV envelope (the M designates Multiple). The major consequence of this novel phenotype is that during primary infection multiple nucleocapsids from an individual ODV enter the same midgut cell. Notably, wild-type AcMNPV ODV particles containing multiple nucleocapsids are much more virulent per os in Trichoplusia ni larvae than ODV particles containing a single nucleocapsid (Washburn et al., 1999). Moreover, studies on AcMNPV pathogenesis have revealed that the onset of BV infection of secondary target cells in several host species occurs much too rapidly to be explained by de novo virus replication (Engelhard et al., 1994
; Flipsen et al., 1995
; Granados & Williams, 1986
; Washburn et al., 1995
, 1998
, 2000
, 2003a
, b
). These studies have provided compelling evidence that AcMNPV (and, by extension, other Group I MNPVs) has evolved a highly unusual infection strategy for exploiting lepidopteran larvae. Specifically, it appears that only a fraction of the nucleocapsids from an ODV particle enters the midgut cell nucleus and uncoats during primary infection, enabling expression of gp64 as an early gene product. At the same time, a separate subpopulation of incoming nucleocapsids migrates to the basal plasma membrane and is repackaged as BV with a membrane containing newly synthesized GP64. This apparently allows the midgut cells infected with AcMNPV ODV to transmit secondary infections to target cells within the host's haemocoel hours before de novo synthesis of BV; this would explain the remarkably rapid onset of secondary infections. Such an infection strategy would give the virus a strong selective advantage because it would obviate one of the principal host defences against baculovirus infection, sloughing ODV-infected midgut cells.
We previously investigated the biological significance of the unusual biphasic mode of GP64 synthesis in H. virescens larvae by comparing the virulence and pathogenesis of AcMNPVs that express gp64 both early and late or only during the late phase of infection. The results of this study were consistent with the nucleocapsid repackaging hypothesis detailed above, as they showed that in H. virescens early GP64 synthesis increased oral virulence and accelerated the onset of ultimately fatal secondary infections within the host's tracheal system (Washburn et al., 2003a). Here we report results from similar studies in which we evaluated the effects of early GP64 synthesis on AcMNPV virulence and pathogenesis in two additional permissive hosts, Spodoptera exigua and T. ni. These insects are significant agricultural pests, and each is from a different subfamily of the Noctuidae (H. virescens Heliothinae; T. ni Plusinae; S. exigua Amphipyrinae). While early GP64 synthesis increased ODV virulence during oral infection of newly moulted fourth instar T. ni larvae, it had no impact on ODV virulence in S. exigua larvae. Furthermore, the absence of any measurable effect of early GP64 synthesis on AcMNPV virulence in this cohort of S. exigua was correlated with two phenomena: (i) remarkably fast and efficient primary infection and (ii) a slow rate of sloughing ODV-infected midgut cells. In contrast, when inoculated 16 h after moulting, early GP64 synthesis was a significant virulence factor in larvae of both species, apparently counteracting the increasing developmental resistance characteristic of older hosts (Engelhard & Volkman, 1995
). Finally, the virus that expressed gp64 in an earlylate, wild-type manner established secondary infections in both hosts hours earlier than the virus that only expressed gp64 during the late phase of infection. These results provide further experimental evidence that early GP64 synthesis is a component of a unique and highly adaptive baculovirus infection strategy for counteracting developmental resistance mechanisms in their insect hosts.
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METHODS |
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Insects.
Eggs of S. exigua were provided by the USDA-ARS Western Cotton Research Laboratory, and T. ni eggs were purchased from Benzon Research (Carlisle, PA, USA). Larvae were reared on synthetic Stoneville diet at 22 or 28±3 °C under constant light through the third instar; under these conditions, both species had five larval instars. Quiescent, late third instar larvae that were preparing to moult were sequestered and observed carefully to determine the exact time of moulting for each insect. In some cases, third instar larvae were held at 7 °C from several hours to overnight in order to regulate their developmental rates and to make large numbers of test insects of the same age available for experiments (Washburn et al., 1995).
Bioassays and time-course experiments.
To establish oral dosemortality relationships for AcCtlNt and Ac21/20, bioassays were done using two developmental cohorts of S. exigua and T. ni. These cohorts were newly moulted and 16 h post-moult fourth instar larvae, hereafter designated 40 and 416, respectively. Individual larvae were inoculated using a Burkhard microinjector with a 1 cm3 tuberculin syringe fitted with a 32 gauge blunt-tipped needle. The needle was inserted through the mouth and into the midgut lumen where the occlusion suspension (0·25 to 1·0 µl in volume) was discharged. Virulence of the BV phenotypes was determined by intrahaemocoelic inoculation of cohorts of 24 h post-moult fourth instar (424) larvae. For these assays, a sharp-tipped 32 gauge needle was inserted through one of the prolegs, and the viral suspension (1 µl volume) was discharged into the larval haemocoel (Engelhard & Volkman, 1995; Engelhard et al., 1994
; Washburn et al., 1995
). Between 26 and 32 insects were used for each assay. After inoculation, test larvae were maintained individually in 25 ml plastic cups containing diet ad libitum in a growth chamber at 28±2 °C until pupation or death from polyhedrosis disease. Viral death was confirmed by microscopic examination (400x) of cadaver tissues for occlusions. Dosemortality relationships for each virushost treatment were evaluated with linear regression using the least squares method.
To investigate the pathogenesis of AcCtlNt and Ac21/20, a series of time-course experiments was carried out using 40 larvae. For these assays, we utilized dosages (determined from bioassays described above) that yielded final mortalities of 90 %. S. exigua were inoculated with 10 occlusions of either recombinant, and T. ni were inoculated with 21 or 30 occlusions of either AcCtlNt or Ac21/20, respectively. At various times during the first 24 h post-inoculation (p.i.), cohorts of between 26 and 32 larvae of each species from the two viral treatments were dissected, and their midguts and associated tissues removed. These tissues were processed to detect the presence of
-galactosidase and examined using stereo (1050x) and/or compound microscopy (100480x) in order to quantify infection foci and identify infected cell types (Engelhard et al., 1994
; Washburn et al., 1995
, 2001
, 2003a
). For each host species, an additional cohort of 32 insects was inoculated orally with AcCtlNt or Ac21/20 occlusions to confirm that each dosage was an LD90.
In a separate experiment, we quantified retention of ODV-infected midgut cells through the moult to the fifth instar using vAc64z. This recombinant cannot synthesize GP64 and can only establish primary (midgut) infections following oral inoculation with occlusions (Monsma et al., 1996). A dosage of 175 occlusions (i.e. sufficient to generate large numbers of primary foci) was used to infect cohorts of 40 and 416 S. exigua and T. ni larvae for time-course experiments similar to those described above.
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RESULTS |
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The developmental resistance acquired by T. ni during the fourth instar was shown previously to result from the reduced ability of AcMNPV to establish and/or maintain primary infections within the midgut (Engelhard & Volkman, 1995; Washburn et al., 1998
). To determine if the basis for developmental resistance was the same in S. exigua, we orally inoculated 40 and 416 larvae with 10 occlusions of AcCtlNt (n=28 for each treatment) and used
-galactosidase as a reporter to quantify viral foci at 12 h p.i. Relative to insects inoculated immediately after moulting, 57 % fewer foci were observed in 416 larvae [40=20·3±4·7 (mean±1 SE) foci; 416=8·8±2·9 foci], which was consistent with the fact that the inoculum had to be doubled to achieve an LD50. Moreover, the decrease in foci number suggested that S. exigua and T. ni shared a similar physiological basis for developmental resistance to AcMNPV.
In contrast to the differences in oral virulence between AcCtlNt and Ac21/20 shown in Fig. 1, these viruses were equally and highly virulent when the midgut was bypassed by injecting BV directly into the host's haemocoel. Following injection into 424 S. exigua, 0·13 p.f.u. of AcCtlNt and Ac21/20 yielded final mortalities of 19 and 11 %, respectively, and dosages of 3·3 p.f.u. yielded final mortalities of 81 and 82 %, respectively. Both viruses provided similar results in T. ni. For example, following inoculation of 424 T. ni larvae with 3·3 p.f.u. of AcCtlNt, 83 % of the larvae succumbed to polyhedrosis disease. These findings demonstrated that early GP64 synthesis was only important for virulence via the natural route of infection (per os). The fact that both hosts were equally susceptible to injection of minute quantities of BV underscored the importance for AcMNPV to establish even a single foothold of infection within the haemocoel.
The results of the time-course experiments in which AcCtlNt and Ac21/20 pathogenesis in S. exigua and T. ni larvae was monitored with the lacZ reporter are shown in Figs 27. As expected, differences in the temporal pattern of gp64 expression did not affect the onset of viral gene expression in ODV-infected midgut cells of either host (Fig. 2
A, B; Washburn et al., 2003a
). With the dosages used in these experiments (LD90),
-galactosidase, which is indicative of early viral gene expression, was detected at the same times after infection of each species by either virus (4 h p.i. in S. exigua and 6 h p.i. in T. ni; Figs 2A, B
). Consistent with the earlier onset of lacZ expression in S. exigua, the rates of primary infection by both viruses were also much greater in S. exigua than in T. ni. Remarkably, with both viruses, the proportion of S. exigua larvae positive for LacZ was predictive of the final mortality as early as 8 to 12 h p.i. By contrast, the time point predictive of final mortality for AcCtlNt-infected T. ni was 24 h p.i. (Fig. 2A
), 12 to 16 h later. Moreover, only 75 % of the cohort inoculated with Ac21/20 was
-galactosidase-positive at 24 h p.i. (Fig. 2B
), indicating that 15 % of the larvae that would ultimately die were not yet expressing lacZ. These results suggested that the severity of developmental resistance was correlated with the rate at which the virus was able to establish gene expression in midgut cells.
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Due to the extreme sensitivity of these permissive hosts to the presence of BV within the haemocoel, the rates at which primary midgut foci established systemic infection, within even a single tracheal cell, accurately reflected the rates at which mortal infections were established within larval cohorts. Because the ODVs of both recombinants infected midgut cells of S. exigua more quickly and in greater numbers than in T. ni (Figs 2, 3), the proportions of S. exigua systemically infected by AcCtlNt and Ac21/20 rose much more rapidly than in the corresponding T. ni cohorts (Fig. 5
A, B). This further explains why the percentages of
-galactosidase-positive S. exigua in both viral treatments were predictive of the final mortality many hours before T. ni.
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To quantify retention rates of ODV-infected midgut cells in S. exigua during larval moulting, another time-course experiment was conducted in which we orally inoculated 40 and 416 S. exigua with 175 vAc64z occlusions. This virus cannot synthesize GP64, and following oral inoculation of host larvae, only midgut infections ensue, facilitating rapid and accurate quantification of primary foci (Monsma et al., 1996). Larvae in each S. exigua cohort were divided into two groups, dissected and examined for lacZ signals in midgut cells after reaching either late fourth or early fifth instar stages (40-inoculated 40 and 48 h p.i.; 416-inoculated 24 and 32 h p.i., respectively). The 40 larvae had 50·5±7·8 and 13·4±3·1
-galactosidase-positive midgut cells in the fourth and fifth instar cohorts, respectively, and the 416 larvae had 56·5±11·2 and 43·2±6·4
-galactosidase-positive cells, respectively. Thus, 26 and 77 % of the ODV-infected primary foci produced during infection of 40 and 416 S. exigua larvae and still present by the end of the fourth instar were retained through the moult to the fifth instar. When we repeated this experiment with the same fourth instar developmental cohorts of T. ni, no midgut cells infected by vAc64z were detected in any fifth instar larva.
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DISCUSSION |
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The earliest detectable expression of lacZ in S. exigua midgut cells was 4 h. This was the same time required for Helicoverpa zea SNPV-hsp70/lacZ (HzSNPV-hsp70/lacZ) to express lacZ in fourth-instar H. zea and H. virescens larvae, and HzSNPV is considered to be the most virulent baculovirus for heliothines (Washburn et al., 2001). By comparison, the first detectable expression of lacZ by both AcMNPV AcCtlNt and Ac21/20 was 6 h in T. ni and 8 h in H. virescens (Washburn et al., 2003a
). The corresponding LD50 values of AcCtlNt in S. exigua, T. ni and H. virescens were 4, 6 and 6 occlusions, respectively, in insects inoculated at 40 and 8, 12 and 18 occlusions in insects inoculated as 416 (Washburn et al., 2003a
). Thus, both the rapidity of early viral gene expression (as indicated by first expression of lacZ in the midgut) and the attenuated sloughing of infected midgut cells in S. exigua enhanced the oral virulence of AcMNPV. The earlier onset of lacZ expression in the midgut and the reduced rate of infected midgut cell sloughing are not independent factors; however, the rate of sloughing increases progressively with time within an instar. Infected midgut cell sloughing exerts strong selection pressure on the virus to transmit infection rapidly to secondary target cells, and early expression of gp64 facilitates this process. Early expression of gp64 had no effect on the timing of lacZ expression in midgut cells. The cost to AcMNPV in terms of increased inoculum required to establish an LD50 with virus that cannot express gp64 early depended on relative rates of sloughing. Hence, in 416 larvae, 4·4, 9·4, and 43·3 times more Ac21/20 was required to achieve an LD50 in S. exigua, T. ni and H. virescens, respectively, relative to AcCtlNt. Early expression of gp64, therefore, is an effective viral mechanism for overcoming developmental resistance, and the earlier the expression, the more effective it is.
Previously, Flipsen et al. (1995) constructed an AcMNPV double reporter recombinant in which lacZ was placed under the control of the functionally early Drosophila melanogaster hsp70 promoter, and
-glucuronidase (
-GUS) was placed under the control of the very late AcMNPV p10 promoter. They used this recombinant to study early pathogenesis in S. exigua larvae and found that
-galactosidase appeared in secondary target cells (undifferentiated midgut epithelial cells) before
-GUS was detected in ODV-infected midgut columnar cells. These results indicated that secondary targets were infected before virus replication and late gene expression had occurred in primary targets. Similarly, with wild-type GP64 synthesis, the lag time between the onset of lacZ expression within infected midgut and tracheal cells of T. ni and S. exigua was only 34 h. We previously reported similar results during the early stages of AcMNPV infection in the semi-permissive hosts Manduca sexta and H. zea (Washburn et al., 1996
, 2000
). In cultured insect cells, it takes
1012 h for the de novo synthesis of AcMNPV BV. Thus, the results from these in vivo studies support the hypothesis that the earliest systemic infections by wild-type AcMNPV arise from ODV-derived nucleocapsids repackaged as BV via early GP64 synthesis. This repackaging phenomenon can only occur with NPVs having the M phenotype. The restriction of the MNPVs to species within the Lepidoptera suggests that evolutionary coupling of these two phenotypic traits has allowed these viruses to exploit larval lepidopterans by overcoming their first, and often only, line of defence, sloughing of infected midgut cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 30 October 2003;
accepted 11 December 2003.