Autographa californica M nucleopolyhedrovirus early GP64 synthesis mitigates developmental resistance in orally infected noctuid hosts

Ji-Hong Zhang1,2, Jan O. Washburn1, Donald L. Jarvis3 and Loy E. Volkman1

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The unusual early synthesis of the Autographa californica M nucleopolyhedrovirus (AcMNPV) budded virus (BV) structural protein GP64 is an important virulence factor during oral infection of Heliothis virescens larvae. Considering the breadth of the AcMNPV host range, the importance of early GP64 synthesis in orally infected permissive hosts (Trichoplusia ni and Spodoptera exigua) from subfamilies other than that of H. virescens was assessed. An AcMNPV recombinant, having wild-type early and late GP64 synthesis, was compared with one in which only late GP64 synthesis occurred. Early GP64 synthesis was found to have more of an effect on virulence in orally inoculated T. ni than S. exigua and that virulence was dependent on two factors: the ability of the host to slough occlusion-derived virus (ODV)-infected midgut cells and the rapidity with which BV was transmitted to the tracheal cells. In both host species, insects inoculated orally with the control virus transmitted BV to tracheal cells hours before those inoculated with the gp64 temporal mutant. Moreover, with early GP64 synthesis, the lag between the onset of viral gene expression in midgut and tracheal cells was only 3–4 h, supporting the conclusion that in these insects, the first systemic infections arose from ODV-derived nucleocapsids repackaged as BV. These results provide further empirical proof that early GP64 synthesis is a component of a unique and selectively advantageous baculovirus infection strategy for exploiting larval lepidopterans by counteracting developmental resistance.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Autographa californica M nucleopolyhedrovirus (AcMNPV) (Baculoviridae: Nucleopolyhedrovirus) is a promising microbial agent for the control of lepidopteran pests, in part because it can fatally infect the larvae of more than 30 species in 12 families, including significant crop pests in the Noctuidae, the most specious of the lepidopteran families (Adams & McClintock, 1991; Granados & Williams, 1986). Nucleopolyhedroviruses (NPVs) require two phenotypes to complete their infection cycles in vivo. The first form, occlusion-derived virus (ODV), is embedded in a crystalline matrix of polyhedrin and initiates primary infection in the host's midgut epithelium following ingestion and dissolution of the viral occlusions. The second form, budded virus (BV), subsequently disseminates the infection throughout the host until the insect succumbs and liquefies, releasing millions of viral occlusions that can then transmit infection to new hosts.

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 early–late, 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus preparation and quantification.
Construction of two AcMNPV recombinants, AcCtlNt-64HB and AcLate21/20-64HB, used in this study has been described previously (Washburn et al., 2003a). AcCtlNt-64HB (hereafter AcCtlNt) has wild-type gp64 expression (early and late), and AcLate21/20-64HB (hereafter Ac21/20) expresses gp64 only during the late phase of infection, coincident with the expression of other viral structural genes. Both viruses contained the Escherichia coli {beta}-galactosidase gene (lacZ) under the control of a Drosophila heat shock promoter (hsp70), which allowed detection of infected cells by the presence of the blue reporter signal (Engelhard et al., 1994). Occlusions and BV of both viruses were harvested at 96 h p.i. from cultured Sf-9 cells. Occlusions were further purified by sucrose gradient centrifugation, suspended in a neutrally buoyant solution of glycerin and water (3 : 2, v/v), quantified using a haemocytometer and stored at 4 °C until used. BV was titrated by plaque assay on Sf-9 cells (Volkman & Goldsmith, 1981) and held at 4 °C until used. A third recombinant virus, vAc64z, which was kindly provided by Gary Blissard (Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, NY, USA) also carries the hsp70/lacZ marker but is unable to synthesize any GP64 (Monsma et al., 1996). vAc64z was used to quantify the retention of ODV-infected midgut cells during moulting.

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 dose–mortality 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. Dose–mortality relationships for each virus–host 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 {beta}-galactosidase and examined using stereo (10–50x) and/or compound microscopy (100–480x) 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.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The dose–mortality relationships for fourth instar larvae of S. exigua and T. ni inoculated orally with occlusions of either AcCtlNt (control) or Ac21/20 (expresses only late GP64 ) are presented in Fig. 1. Both developmental cohorts of fourth instar S. exigua were more susceptible to fatal infection by AcCtlNt and Ac21/20 than the corresponding cohorts of T. ni. Even so, the 40-inoculated larvae of both species were highly susceptible; a single AcCtlNt occlusion generated 21·9 and 12·5 % larval mortality in S. exigua and T. ni, respectively (Fig. 1A). Unexpectedly, newly moulted S. exigua had virtually identical susceptibility to AcCtlNt and Ac21/20 (LD50s of 4 occlusions; LD90s of 10 and 12 occlusions for AcCtlNt and Ac21/20, respectively; Fig. 1A, B), demonstrating that for this developmental stage early GP64 synthesis was inconsequential to oral virulence. In contrast, the AcCtlNt LD50 for 416 S. exigua was 8 occlusions, demonstrating that even in this extremely susceptible host, a twofold increase in wild-type viral inoculum was required to overcome the developmental resistance acquired during the first 16 h after moulting. For Ac21/20, however, an 8·75-fold increase in inoculum (35 occlusions) was needed to achieve an LD50 in 416 S. exigua. Hence, early gp64 expression provided a 4·4-fold increase in the ability of AcMNPV to overcome the developmental resistance acquired by the older larvae.



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Fig. 1. Dose–mortality relationships for S. exigua (Se) and T. ni (Tn) larvae inoculated orally as newly moulted fourth instars (A and B, respectively) or 16 h after the moult (C and D, respectively) with occlusions of either AcCtlNt or Ac21/20. Each point represents the percentage of a cohort containing between 22 and 32 insects that died from baculovirus infection while in the larval stage.

 
In T. ni, early gp64 expression had an even greater impact on virulence. First, unlike the 40 S. exigua, in which early GP64 synthesis was inconsequential, 1·8-fold more Ac21/20 occlusions were required for an LD50 in T. ni, as compared to AcCtlNt (11 and 6 occlusions, respectively). This shows that following oral inoculation of newly moulted larvae, early GP64 synthesis reduced by almost half the amount of inoculum needed for an LD50 and therefore significantly increased virulence. In 416 T. ni, the cost of not expressing gp64 during the early phase of infection was even greater, as 9·4-fold more inoculum was required for an LD50 in this cohort compared to 416 insects inoculated with AcCtlNt (113 vs 12 occlusions). These results showed that T. ni has greater developmental resistance to AcMNPV infection than S. exigua, and that the ability of early GP64 synthesis to enhance virulence was proportional to the degree of developmental resistance (Washburn et al., 2003a).

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 {beta}-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 2–7. 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. 2A, B; Washburn et al., 2003a). With the dosages used in these experiments (LD90), {beta}-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 {beta}-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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Fig. 2. Percentages of S. exigua and T. ni larvae positive for LacZ at various hours after inoculation as newly moulted fourth instars with either AcCtlNt (A) or Ac21/20 (B). S. exigua were inoculated with 10 occlusions of either AcCtlNt or Ac21/20, and T. ni larvae were inoculated with 21 or 30 occlusions, respectively, of either AcCtlNt or Ac21/20. All doses yielded a final larval mortality level of ~90 % (Fig. 1). Each point represents the proportion of LacZ-positive larvae from a cohort of between 26 and 32 larvae.

 
The temporal pattern of viral focus formation was consistent with the greater virulence of both viruses in S. exigua than in T. ni. Despite being inoculated with fewer occlusions, both viruses produced more numerous and persistent primary foci in S. exigua than in T. ni (Fig. 3A, B). After controlling for dosage, each AcCtlNt occlusion produced a mean of 2·5 foci in S. exigua, as compared to only 0·4 foci in T. ni, and the ratio of focus formation for Ac21/20 was 2·5 : 0·6. Thus, these viruses produced six- and four-fold more foci in S. exigua than in T. ni, respectively. Moreover, foci numbers rose more slowly in T. ni larvae, peaked at 15 h p.i. and then abruptly declined (Fig. 3A, B; Fig. 4, Washburn et al., 1999). At 18 h p.i., the numbers of foci observed in AcCtlNt and Ac21/20-infected T. ni larvae were reduced by 66 and 83 % as compared to 15 h p.i., suggesting that many ODV-infected midgut cells had been sloughed. The observation that more Ac21/20 than AcCtlNt foci were required to achieve an LD90 in T. ni showed that the ODV of the latter was more efficient at establishing systemic infections.



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Fig. 3. Mean numbers of viral foci (±1 SE) in S. exigua and T. ni larvae at various hours after inoculation as newly moulted fourth instars with either AcCtlNt (A) or Ac21/20 (B). S. exigua larvae were inoculated with 10 occlusions of either AcCtlNt or Ac21/20, and T. ni larvae were inoculated with 21 or 30 occlusions, respectively, of either AcCtlNt or Ac21/20. Each point represents the mean foci numbers in LacZ-positive larvae from a cohort of between 26 and 32 larvae. Due to the confluence of viral plaques, Ac21/20 foci for T. ni sampled at 24 h p.i. could not be quantified.

 


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Fig. 4. Percentages of AcCtlNt (A) and Ac21/20 (B) foci with one or more LacZ-positive tracheal cells in S. exigua and T. ni at various times after inoculation as newly moulted fourth instars. S. exigua larvae were inoculated with 10 occlusions of either AcCtlNt or Ac21/20, and T. ni larvae were inoculated with 21 or 30 occlusions, respectively, of either AcCtlNt or Ac21/20. Each point represents mean foci numbers in LacZ-positive larvae from a cohort of between 26 and 32 larvae. Regression lines for S. exigua (Se) are solid, and those for T. ni (Tn) are dashed.

 
During the early stages of viral pathogenesis, the primary midgut cellular targets of AcCtlNt and Ac21/20 in both S. exigua and T. ni were mature columnar epithelial cells. Similarly, the second cell type expressing lacZ in both hosts was tracheolar cells servicing ODV-infected midgut cells; the proximity of primary and immediate secondary targets indicated that these tracheolar cells were infected directly by BV generated from the ODV-infected midgut cells. Infection of tracheolar cells signalled the onset of irreversible fatal systemic infections, and for all four virus–host combinations, {beta}-galactosidase signals within the tracheal epidermis rose linearly over time and at virtually identical rates [i.e. regression coefficients (slopes) of 3·8–4·1, Fig. 4A, B]. While the BVs of both viruses established secondary infection at similar rates, the onset by Ac21/20 was delayed by several hours relative to AcCtlNt. Using the linear equations correlating the prevalence of tracheal cell-containing viral foci with hours p.i. (Fig. 4A, B), the predicted times for the onset of tracheal infection of S. exigua larvae by AcCtlNt and Ac21/20 BV were 4·2 and 9·6 h p.i., respectively. In T. ni larvae the times predicted for the onset of tracheal infections were 5·7 and 10·4 h p.i. Thus, early GP64 synthesis accelerated establishment of systemic infection by 5·4 h in S. exigua and by 4·7 h in T. ni.

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. 5A, B). This further explains why the percentages of {beta}-galactosidase-positive S. exigua in both viral treatments were predictive of the final mortality many hours before T. ni.



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Fig. 5. Percentages of S. exigua and T. ni larvae with LacZ expression in the tracheal epidermis at various times after inoculation as newly moulted fourth instars with either AcNtlCt (A) or Ac21/20 (B). S. exigua larvae were inoculated with 10 occlusions of either AcCtlNt or Ac21/20, and T. ni larvae were inoculated with 21 or 30 occlusions, respectively, of either AcCtlNt or Ac21/20. Each point represents the proportion of larvae from a cohort of between 26 and 32 larvae. Regression lines for S. exigua (Se) are solid, and those for T. ni (Tn) are dashed.

 
In addition to temporal changes in foci numbers (Fig. 3A, B), the cellular composition of AcCtlNt and Ac21/20 foci provided further evidence that T. ni larvae sloughed infected midgut cells more frequently than S. exigua. During the early stages of AcMNPV pathogenesis in vivo, three kinds of viral foci can be distinguished: (i) midgut restricted, in which BV transmission to secondary targets has not occurred (Fig. 6A); (ii) midgut and tracheal, in which BV has been transmitted and the primary target remains (Fig. 6B); and (iii) tracheal, in which BV transmission has occurred and the primary target has been sloughed (Fig. 6D, C) (Washburn et al., 1999, 2001, 2003a, b). Because tracheal epidermal cells are not exposed to the midgut lumen, they cannot be infected directly by ODV. Therefore, analysis of foci restricted to the tracheal epidermis provides a way to measure the rates at which infected larvae sloughed ODV-infected midgut cells. In our experiments with all four host–virus combinations, the temporal increases in the proportions of such foci were well described by linear models (Fig. 7A, B). For the two larval cohorts in each viral treatment, the ratio of the regression coefficients provided an estimate for the relative rates at which the primary midgut cellular targets infected by each virus were shed by S. exigua and T. ni larvae. These calculations indicated that T. ni larvae sloughed midgut cells infected by the ODVs of AcCtlNt and Ac21/20 at rates that were 4·8 and 14·9 times greater than S. exigua, respectively (Fig. 7A, B).



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Fig. 6. Cellular composition of viral foci during early pathogenesis of AcCtlNt in S. exigua orally inoculated as newly moulted fourth instars. (A) Viral focus consisting of a single LacZ-positive columnar cell (M), the primary cellular target of ODV at ~18 h p.i. (B) Multicellular viral focus consisting of a LacZ-positive, ODV-infected columnar cell (M) and a BV-infected tracheolar cell (T) at ~18 h p.i. Tracheolar cells are the immediate targets of BV produced by ODV-infected midgut cells. (C) A LacZ-positive columnar cell (M) in the process of being sloughed from the midgut epithelium. Note the rounded apical cell body and the constricted cytoplasmic strand anchoring the cell to the midgut basement membrane. The image was captured from a newly moulted fifth instar insect ~44 h after inoculation. (D) Multicellular viral focus restricted to the tracheal epidermis and consisting of two LacZ-positive tracheolar cells (T) at ~24 h p.i. The underlying ODV-infected columnar cell presumably has been sloughed from the midgut epithelium. The bar in each panel is equal to 40 µm.

 


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Fig. 7. Percentages of AcCtlNt (A) and Ac21/20 (B) viral foci restricted to the tracheal epidermis of S. exigua and T. ni larvae at various times after inoculation. S. exigua larvae were inoculated with 10 occlusions of either AcCtlNt or Ac21/20, and T. ni larvae were inoculated with 21 or 30 occlusions, respectively, of either AcCtlNt or Ac21/20. Each point represents the proportion of larvae from a cohort of between 26 and 32 larvae. Regression lines for S. exigua (Se) are solid, and those for T. ni (Tn) are dashed.

 
Notably, in our time-course experiments, some fifth instar S. exigua orally inoculated during the fourth instar were found to harbour {beta}-galactosidase-positive midgut cells, implying that ODV-infected primary cellular targets in this host could be retained through the moult (Fig. 6C). This phenomenon was not observed in previous studies of AcMNPV-hsp70/lacZ pathogenesis in T. ni and H. virescens larvae because these species clear their midgut tissues of all ODV-infected cells during the first moult after inoculation (Washburn et al., 1995). To determine if the midgut cell infections in fifth instar S. exigua could have resulted from BV transmission from infected tracheolar cells, we inoculated 255 p.f.u. of AcCtlNt BV intrahaemocoelically into feeding fourth instar larvae (n=6), killed the larvae at 24 h p.i., and examined their tissues for lacZ signals. While numerous {beta}-galactosidase-positive cells were found within the tracheal epidermis on the distal side of the midgut basal lamina, no {beta}-galactosidase-positive midgut epithelial cells were found, suggesting that midgut cells were not infected by BV from the haemocoel.

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 {beta}-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 {beta}-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.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results of the experiments reported here demonstrated that early expression of the AcMNPV BV structural protein GP64 was an important virulence factor during oral, but not intrahaemocoelic, infection of fourth instar larvae of the noctuids T. ni and S. exigua. The effect of early GP64 synthesis on AcMNPV virulence, however, varied significantly with species and developmental cohort. Early GP64 synthesis did not measurably enhance ODV virulence for newly moulted S. exigua larvae, whereas it strongly enhanced virulence for T. ni inoculated 16 h after moulting (Fig. 1). Overall, S. exigua larvae were more susceptible than T. ni to mortal infection by the ODVs expressing gp64 in wild-type fashion (early–late) or only during the late phase of infection. The extreme oral susceptibility of S. exigua was attributed to three separate, but linked, phenomena: (i) the establishment of more foci per occlusion; (ii) the rapid rate at which primary cellular targets were infected; and (iii) the inability of larvae to shed ODV-infected midgut cells. Indeed, the sloughing response of fourth instar S. exigua was so weak that significant numbers of ODV-infected midgut cells were retained for as long as 48 h p.i., and in some cases even after larvae had moulted to the fifth and final larval instar.

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 {beta}-glucuronidase ({beta}-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 {beta}-galactosidase appeared in secondary target cells (undifferentiated midgut epithelial cells) before {beta}-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 3–4 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 ~10–12 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.


   ACKNOWLEDGEMENTS
 
Financial support was provided by USDA NRICG 95-37302-1921, NSF IBN0090632, the Torrey Mesa Research Institute and Syngenta Research and Technology. The China Scholarship Council and the innovation project of the Chinese Academy of Sciences, KSCX2-SW-105, provided partial support for Ji-Hong Zhang. We are grateful to Gary Blissard for providing occlusions of vAc64z, and to USDA-ARS for providing S. exigua eggs. We thank Ian Lin, Eric Chan, Taro Ohkawa and Eric Haas-Stapleton for technical assistance and Steven Ruzin of the CNR Biological Imaging Facility for assistance with the photographs in Fig. 6.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 30 October 2003; accepted 11 December 2003.