INTRODUCTION

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Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the type species of the Nucleopolyhedrovirus genus of the family Baculoviridae. Baculoviruses are arthropod specific and generally produce fatal infections in their hosts. These pathogens are characterized by rod-shaped, enveloped nucleocapsids that contain supercoiled, circular, double-stranded DNA. The infection cycle of AcMNPV and many other baculoviruses includes two distinct phenotypes, an occluded form (occlusion-derived virus [ODV]) and a budded form (budded virus [BV]). ODV is embedded within a crystalline protein structure (the occlusion) which helps protect the infectious virions from environmental degradation. In the nucleopolyhedroviruses, ODV is composed of virions containing either a single nucleocapsid (SNPVs) or one to many nucleocapsids (MNPVs).

The ODV and BV phenotypes of AcMNPV play unique roles in the viral life cycle; ODV spreads infection among individual hosts, whereas BV transmits infection from cell to cell within a host. In larval lepidopterans, the hosts of AcMNPV, ODV is released after ingestion by the highly alkaline digestive juices of the midgut, which dissolve the occlusions (11). Infections are initiated when the ODV envelope fuses with the plasma membrane of a microvillus on a columnar epithelial cell that is exposed to the midgut lumen (12). The nucleocapsids subsequently enter the cell and move through the cytoplasm to the nucleus, which they enter via nuclear pores (11, 15, 25). During viral replication, BV buds from the basal plasma membrane as a single nucleocapsid that gains a loose-fitting envelope in the process (26, 27). The BV envelope contains spikes of gp64, a virally encoded protein that is essential for the infection to progress beyond the midgut in Trichoplusia ni (16). BV spreads infection among the host tissues until nearly the entire insect is infected, and in the final stages of pathogenesis, the host dies and liquefies in a dramatic "meltdown," which releases millions of occlusions that can initiate infections when ingested by new hosts.

While a few NPVs are known to infect decapod crustaceans (3), the majority of described species have been isolated from insects, mostly Lepidoptera (28). The SNPVs also have been isolated from members of the Diptera, Hymenoptera, and Trichoptera, while the MNPVs have been isolated only from members of the Lepidoptera, the youngest of the insect orders (17). The restriction of the MNPVs to the Lepidoptera suggests that these viruses originated from the SNPVs and then radiated along with their hosts through evolutionary time (17). In this context, it is intriguing that the MNPVs have evolved the life history trait of packaging multiple nucleocapsids within a single envelope. Such a strategy might seem inefficient in vivo because fatal infections can be initiated within primary target cells of the host midgut epithelium by virions containing single nucleocapsids, as evidenced by the SNPVs. In addition to apparently being wasteful of infectious units, the multiple-nucleocapsid packaging strategy results in larger virions, which could pose physical constraints on movement through the peritrophic membrane of the midgut and/or the narrow microvilli of primary target cells (27). Why, then, would natural selection favor a redundant delivery system in which multiple nucleocapsids infect the same cell?

At least two hypotheses have been proposed to explain the multiple-nucleocapsid-per-virion packaging strategy of the MNPVs. First, it is well documented that viral occlusions rapidly lose viability when exposed to sunlight, presumably because of UV radiation damage to the viral DNA (8). By infecting primary target cells with multiple copies of the viral genome, it is possible that gene complementation compensates for damaged DNA and facilitates productive infections (17). The second hypothesis postulates that infection of primary target cells with multiple copies of the viral genome increases infection efficiency in vivo by accelerating the onset of secondary infection (1, 27). In this scenario, when multiple nucleocapsids from a single ODV particle infect the same cell, some of the nucleocapsids enter the nucleus and others remain in the cytoplasm. The viral genome(s) within the nucleus initiates gene expression and replication, whereas nucleocapsids that do not penetrate the nucleus are repackaged as BV and directly infect secondary target cells before viral replication is completed within the primary target.

While these two hypotheses are not mutually exclusive, several features of the baculovirus life history, as well as empirical evidence from studies of AcMNPV pathogenesis in vivo, are consistent with the second scenario. First, gp64 is unusual among viral structural proteins in that its synthesis is regulated by both an early and a late promoter (2, 14), allowing this essential glycoprotein to be produced and integrated into the plasma membrane of primary target cells hours before the de novo synthesis of progeny BV. Thus, AcMNPV has a built-in genetic mechanism for repackaging ODV. Second, in time course experiments aimed at elucidating the early events of AcMNPV pathogenesis in Spodoptera exigua, infection of secondary target cells, in this case midgut regenerative cells, was evident before late and very late gene expression had occurred in primary target cells, suggesting that regenerative cell infection had been mediated by repackaged parental nucleocapsids (9). Third, the loss of infected midgut cells is a key mechanism underlying developmental resistance in susceptible hosts such as T. ni and Heliothis virescens (7, 31), implying that sloughing primary target cells may be a common host response to baculovirus infection and one that might function as a powerful selection force for the rapid establishment of systemic infections.

To investigate the biological significance of the multiple nucleocapsid packaging of MNPVs in vivo, we isolated ODV fractions of AcMNPV carrying a -galactosidase reporter gene (AcMNPV-hsp70/lacZ) enriched for virions containing either single (ODV-S) or multiple (ODV-M) nucleocapsids. We used these two inocula in time course experiments to compare the early events of viral pathogenesis, particularly the temporal onset of systemic infections, in fourth-instar T. ni. Our hypothesis was that if the nucleocapsids of ODV-M actually are repackaged as BV, insects challenged with this inoculum should exhibit increased rates of systemic infection than those challenged with ODV-S. The results demonstrated clearly that infections initiated by ODV-M moved more rapidly into secondary target cells of the tracheal epidermis of T. ni than did infections initiated by ODV-S. In addition, comparable mortality levels were achieved by ODV-M with far fewer primary midgut foci than those generated by ODV-S, implying that ODV-M was more efficient at establishing systemic infections.

	MATERIALS AND METHODS

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Virus preparation and assessment. Occlusions of AcMNPV-hsp70/lacZ (5) used in this study were isolated from liquefied cadavers of T. ni and partially purified by centrifugation on sucrose gradients (22). ODV was subsequently liberated by exposure of occlusions to dilute alkaline saline (0.1 M Na₂CO₃, 0.1 M NaCl) and then banded by density equilibrium centrifugation on 25 to 59% sucrose gradients. The pale gray-blue bands of virus were collected individually with a 5-ml syringe and a 22-gauge needle. Samples from each fraction were negatively stained with 2% uranyl acetate and viewed by transmission electron microscopy (TEM) (JEOL model 100 CX). Fractions enriched with virions containing single nucleocapsids were identified and pooled (ODV-S), as were fractions enriched with virions containing multiple nucleocapsids (ODV-M). We examined over 500 negatively stained virions from both the ODV-S and ODV-M stock solutions by TEM to determine the distribution of nucleocapsids per virion. These stock solutions, aliquoted into Eppendorf tubes and stored at 80°C, were diluted to the appropriate concentration with Grace's medium (JRH) plus 1% fetal bovine serum (Gemini Bio-products, Inc.) prior to use.

Viral protein and genome quantification. Viral protein concentrations were estimated by the Bradford protein assay (Bio-Rad). Genomic copy numbers were estimated by Southern hybridization; known concentrations of pAcEcoRI-I DNA (4) and various dilutions of viral DNA were hybridized to a radiolabeled probe, and the signals were compared. The plasmid DNA was used to generate a standard curve because its concentration could be accurately determined by optical density, its size is known, and it contains a unique region of the AcMNPV genome. Equivalent copy numbers of viral genome and plasmid DNA should therefore register the same level of signal, and the plasmid copy number (and, by extrapolation, viral genome copy number) could be calculated for a given concentration of plasmid DNA determined by measuring the optical density at 260 nm. Experimentally, DNA was isolated from a known volume of our stock samples of ODV-S and ODV-M by proteinase K digestion followed by phenol-chloroform extraction. Both viral DNA and plasmid DNA were digested with BamHI, releasing a 927-bp fragment containing the AcMNPV open reading frame 1629 and polyhedrin genes. Dilutions of the digested plasmid and viral DNA were electrophoresed in a 1.2% agarose gel in Tris-acetate-EDTA (TAE) buffer and then transferred to a Nytran membrane. The BamHI fragment excised from pAcEcoRI-I also was used as the probe; for this purpose, it was purified from an agarose gel by using the QIAEX DNA gel extraction kit and then labeled with [-³²P]dATP by random priming. Hybridizing probe was quantified with a PhosphorImager (Molecular Dynamics) and ImageQuant software. Determinations of viral genome content for ODV-S and ODV-M were performed three times each, starting with extraction of viral DNA.

Maintenance of test larvae. Eggs of T. ni were purchased from Ecogen Inc. (Langhorne, Pa.), and larval diet (Stoneville) was provided by the American Cyanamid Company (Princeton, N.J.). Larvae were reared in groups under constant light at 28 ± 3°C until the end of the third larval instar. Nonfeeding, quiescent third-instar larvae preparing to molt were culled and stored at various temperatures between 4 and 22°C for 4 to 12 h to synchronize developmental rates, making large numbers of test insects of the same age available for experiments (30).

Bioassay and time course experiments. Virus, in 1.0-µl aliquots, was orally inoculated into the anterior midgut of newly molted (within 15 min of shedding the third-instar cuticle) fourth-instar larvae. The inoculum was delivered via a syringe fitted with a 32-gauge blunt-tip needle that was mounted onto a microapplicator (ISCO or Burkard). After inoculation, the larvae were maintained in a growth chamber at 28 ± 2°C under constant illumination in individual 25-ml plastic cups containing diet. For bioassays, cohorts of 25 or more larvae were inoculated and maintained until death or pupation. For time course experiments, ~70% lethal doses (LD₇₀) of ODV-S and ODV-M (determined from the bioassays) were inoculated into three discrete cohorts of larvae over a 3-week period. For each cohort, separate internal controls (bioassays) were used to confirm that mortality levels were consistent. In total, between 28 and 34 larvae were sacrificed at each of seven time points from 2 to 22 h postinoculation (p.i.). The intact midgut and associated tissues were removed from each larva and processed for lacZ elucidation as previously described (5, 30). The preparations were examined for lacZ expression by stereo (magnification, ×10 to ×50) and/or compound (magnification, ×100 to ×480) microscopy to quantify foci of infection and infected cell types (31). In these studies, several critical benchmarks of infection were established, including the timing and identity of the first host cells expressing lacZ, the onset of systemic infections in the host tracheal system, and the time point at which the proportion of lacZ-expressing insects was predictive of the mortality level obtained in bioassays. The analysis of AcMNPV pathogenesis presented here is based upon the distribution patterns of the reporter gene product among tissues from 199 and 219 insects challenged with the ODV-S and ODV-M inocula, respectively.

	RESULTS

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Characterization of ODV inocula. Examination of the ODV-S and ODV-M stocks by TEM confirmed that the ODV-S stock consisted primarily of virions with single nucleocapsids and that the ODV-M stock consisted exclusively of virions containing two or more nucleocapsids (Fig. 1). Evaluation of 530 virions from the ODV-M pool revealed that 78% contained two to four nucleocapsids per envelope and the balance contained five or more (Fig. 2). In contrast, 86% of the 514 virions evaluated from the ODV-S pool contained virions with only one nucleocapsid while the remaining 14% consisted of virions with two to four nucleocapsids (Fig. 1 and 2). While 14% contamination of the ODV-S stock with ODV-M was not ideal, we reasoned that it should not prohibit the detection of major differences in pathogenesis induced by the two virus populations.

View larger version (85K): [in this window] [in a new window]   FIG. 1.   Electron micrographs of AcMNPV-hsp70/lacZ ODV-M (A) and ODV-S (B) inocula used in this study.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Frequency distribution of the number of nucleocapsids per envelope of the AcMNPV-hsp70/lacZ ODV-S and ODV-M inocula used in this study. The frequencies were determined from counts of nucleocapsids within ODV particles from randomly chosen fields viewed by TEM.

Time course experiment. We found that the proportions of lacZ-positive T. ni larvae sampled at various time points following challenge with ODV-S and ODV-M were very similar (Fig. 3) and that primary infection of larval cohorts from both treatments occurred at comparable rates. lacZ expression was first detected at 2 h p.i. in 3 and 13% of the larvae in the ODV-S- and ODV-M-inoculated cohorts, respectively. In a separate test, no lacZ expression was detected in larvae sampled at 1 h after infection with either ODV preparation administered at dosages approximately 10 times an LD₁₀₀ (data not shown). For both ODV populations, the proportions of infected larvae increased rapidly during the 4 h after first signaling (2 to 6 h p.i.) and, by 6 h p.i., were predictive of the average final mortalities in the companion bioassays (Fig. 3; ODV-S: 62% at 6 h p.i., LD₆₇; ODV-M: 73% at 6 h p.i., LD₆₃). Between 8 and 22 h p.i., the proportions of lacZ-positive larvae fluctuated but remained within ±12% of the final mortalities for ODV-S- and ODV-M-inoculated insects (Fig. 3).

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Proportions of lacZ-positive T. ni larvae at various times after infection with AcMNPV-hsp70/lacZ ODV-S (open squares) or ODV-M (solid squares). Between 28 and 34 larvae were analyzed for each time point, with the exception of ODV-S at 8 h p.i., for which the sample size was 21. The mean percent mortalities from three bioassays for each inoculum conducted concurrently are shown.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Number of foci per infected T. ni larva at various hours after inoculation with AcMNPV-hsp70/lacZ ODV-S (open squares) or ODV-M (solid squares). Between 28 and 34 larvae were analyzed for each time point, with the exception of ODV-S at 8 h p.i., for which the sample size was 21. Bars indicate 1 standard error.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Percentages of foci containing one or more tracheal cells from T. ni inoculated with AcMNPV-hsp70/lacZ ODV-S (open squares) or ODV-M (solid squares) and sampled at various time points after inoculation. A total of 343 ODV-M and 2,910 ODV-S foci were used to calculate the frequencies of tracheal infections. The two lines were constructed by least-squares regression (ODV-S: y = 4.31x 18.81, r2 = 0.90; ODV-M, y = 8.60x  28.81, r2 = 0.94) (see the text for discussion).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Percentages of T. ni larvae with one or more lacZ-positive tracheal cells at various time points after inoculation with ODV-S (open squares) or ODV-M (solid squares). Between 28 and 34 larvae were analyzed for each time point, with the exception of ODV-S at 8 h p.i., for which the sample size was 21.

	DISCUSSION

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Results from our study provide compelling evidence that multiple nucleocapsid packaging of AcMNPV ODV had little effect on primary midgut infection but significantly facilitated secondary infection of tracheal epidermal cells in fourth-instar T. ni. These results are consistent with those of van Beek et al. (24), who found that an AcMNPV ODV fraction containing seven to nine nucleocapsids killed neonatal T. ni larvae significantly faster than an ODV fraction consisting of virions with single nucleocapsids. In our study, to realize mortality comparable to ODV-M in a cohort of T. ni, 4.5 times as many ODV-S particles had to be inoculated (Table 1). This difference in virion content between the ODV-S and ODV-M inocula was reflected by the increased number of primary foci per ODV-S-infected insect observed in time course experiments (Fig. 4). We saw no delay in the onset of lacZ expression in midgut cells infected by ODV-M relative to ODV-S; the larger physical size of ODV-M, therefore, apparently was not a significant impediment to viral passage from the midgut lumen to within the nuclei of primary target cells (27). The frequencies of foci with tracheal involvement at 6 h p.i. and later time points (Fig. 5) indicated that BV emitted from ODV-M-infected midgut cells established systemic infections twice as fast as did BV emitted from ODV-S-infected midgut cells. Moreover, at the last time point sampled before to the molt (22 h p.i.), threefold more ODV-S foci were restricted to the midgut. These data suggested that ODV-S foci probably incurred a much higher failure rate in establishing systemic infections because all infected midgut cells are sloughed before ecdysis to the fifth instar (7, 30). In addition, the decrease in the average number of foci per infected larva and the increase in the frequency of foci consisting exclusively of tracheal cells for both inocula throughout the time course demonstrated that sloughing of infected cells also occurred during the feeding stage of the fourth instar. It is highly unlikely that tracheal cells themselves were primary targets of infection, because these cells do not have direct access to the midgut lumen. Furthermore, ODV envelopes lack gp64, which is essential for infection to be transmitted beyond the midgut in this species (16). While midgut cell sloughing should reduce the efficiency of establishing systemic infections for both ODV-S and ODV-M, the reduction would be proportionately greater for ODV-S owing to its slower movement into the tracheal system.

In 1981, Granados and Lawler (12) published evidence suggesting that parental nucleocapsids of AcMNPV could bypass the columnar cell nucleus and bud directly through the peplomer-studded, basal plasma membrane and thereby establish systemic infection rapidly in T. ni. Subsequently, studies in which reporter gene recombinants of AcMNPV were used to track virus movement among host tissues revealed lags of less than 6 h between primary and secondary infection in larvae of T. ni (5), H. virescens (30, 31), Helicoverpa zea, Manduca sexta (31a), and S. exigua (9) orally infected with viral occlusions or ODV. While we do not know the time required for AcMNPV replication in T. ni midgut cells, the BV produced by T. ni-derived TN-368 cells inoculated with AcMNPV BV at a multiplicity of infection of 20 is not detected until after 10 h p.i. (29). In contrast, ODV-M-initiated infections in our study exhibited only a 4-h lag between the onset of lacZ expression in primary (2 h p.i.) and secondary (6 h p.i.) target cells. We also observed this to occur in 2 of 376 foci (0.5%) in ODV-S-inoculated insects, a finding that was inconsistent with our hypothesis. The most reasonable explanation for this observation, however, is the 14% contamination of the ODV-S inoculum with ODV-M (Fig. 2). Another possible explanation, although highly unlikely, is that the same midgut cell was infected by two or more ODV-S particles. We do not favor this explanation because of the small numbers of lacZ-positive cells in each infected larva (Fig. 4) and the fact that there are over 200,000 potential primary target cells within the midgut epithelium of a single fourth-instar T. ni (6).

Several lines of empirical evidence suggest that sloughing of infected midgut cells is the first line of defense against AcMNPV infection and has therefore been a major force shaping AcMNPV infection strategies. The ability to slough cells with primary infections is the principal mechanism underlying developmental resistance within the fourth instar of highly susceptible hosts such as T. ni and H. virescens (7, 31). Additionally, stilbene optical brighteners (e.g., M2R) that greatly enhance fatal infection by many baculoviruses (both SNPVs and MNPVs) (13, 18-21, 23, 32, 33) improve AcMNPV infectivity specifically by preventing the host from sloughing infected midgut cells (31). Moreover, because M2R also acts as a synergist for pathogens within the Poxviridae and Reoviridae (13, 18, 19), midgut cell sloughing may be a widespread response by lepidopteran larvae to insect viruses in general. Finally, data from this and previous studies show that highly susceptible hosts, such as T. ni and H. virescens, succumb to AcMNPV when BV infects even a single tracheal epidermal cell (see, e.g., references 7 and 30). For AcMNPV, the payoff for circumventing midgut cell sloughing is extremely high, and it is not surprising that this pathogen has evolved complementary traits that facilitate the rapid establishment of irreversible, fatal secondary infections. The packaging of redundant nucleocapsids within a single virion by the MNPVs also may explain why the MNPVs, in general, have broader individual host ranges than the SNPVs (27). However, this strategy would work only if the major surface glycoprotein of the BV phenotype were driven by an early promoter. This character, in fact, has been described for several MNPVs (10).