Effects of Ac150 on virulence and pathogenesis of Autographa californica multiple nucleopolyhedrovirus in noctuid hosts

Ji-Hong Zhang{dagger}, Taro Ohkawa, Jan O. Washburn and Loy E. Volkman

Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA

Correspondence
Loy E. Volkman
lvolkman{at}nature.berkeley.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ac150 is expressed late during infection of cultured lepidopteran insect cells by Autographa californica multiple nucleopolyhedrovirus. The Ac150 gene product is predicted to have a molecular mass of 11 161 Da and consists of a hydrophobic N terminus and a single ‘peritrophin-A’-like domain, connected by a short region of charged amino acids. An Ac150 deletion mutant and its parental wild-type virus were compared for differences in virulence by both oral and intrahaemocoelic routes of infection. It was found that the mutant was significantly less virulent in larvae of all three host species tested (Heliothis virescens, Spodoptera exigua and Trichoplusia ni) when occlusions were administered orally, but not when isolated occlusion-derived virus (ODV) was administered orally or budded virus was administered intrahaemocoelically. ODV yields were the same from equal numbers of mutant and wild-type occlusions, and nucleocapsid-distribution frequencies within the two ODV populations were the same, eliminating these features as explanations for the observed differences in virulence. Comparison of pathogenesis, as revealed by lacZ expression from identical reporter-gene cassettes in the mutant and wild-type virus, indicated that the mutant was less efficient at establishing primary infection in midgut cells; otherwise, it exhibited infection kinetics identical to those of wild-type virus. Ac150, therefore, can be considered a per os infection factor that mediates, but is not essential for, oral infection.

{dagger}Present address: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the genus Nucleopolyhedrovirus, family Baculoviridae, fatally infects the larvae of more than 30 species of Lepidoptera (Adams & McClintock, 1991; Granados & Williams, 1986). Completion of the infection cycle in vivo requires two genetically identical, but morphologically distinct, viral phenotypes, each of which performs a specific role during infection. Occlusion-derived virus (ODV) is embedded within a crystalline matrix of polyhedrin protein that forms an occlusion, a polyhedral structure that dissolves upon exposure to the highly alkaline juices within the host's midgut lumen. ODV is a specialist that only infects columnar epithelial cells of the midgut. The ODV-infected midgut cells produce budded virus (BV), which, in turn, infects tracheolar cells servicing the infected midgut epithelium (Engelhard et al., 1994; Washburn et al., 1995). Tracheolar cells also produce BV, a generalist that spreads infection to other tissues throughout the insect until death and liquefaction ensue, the hallmarks of polyhedrosis disease.

During the early stages of AcMNPV pathogenesis in penultimate larvae of the permissive hosts, Trichoplusia ni, Spodoptera exigua and Heliothis virescens, secondary infection by BV of even a single tracheolar cell leads to overwhelming infection and death (Engelhard & Volkman, 1995; Washburn et al., 1995; Zhang et al., 2004). Host larvae, however, can clear primary infection by sloughing ODV-infected midgut cells, a defensive response that varies qualitatively among host species and temporally within instars of a single species (Inoue & Miyagawa, 1978; Briese, 1986; Keddie et al., 1989; Engelhard & Volkman, 1995; Washburn et al., 1995, 1998, 1999, 2003). If a host can eliminate ODV-infected midgut cells prior to BV transmission to secondary targets, systemic infection fails and the insect survives. The ability to slough infected cells increases as larvae age and this response is an important component of developmental resistance (Engelhard & Volkman, 1995; Washburn et al., 1998). It is not surprising, therefore, that selection has favoured an AcMNPV infection strategy that incorporates both the timely onset of primary midgut infection and the rapid transmission of BV to nearby tracheolar cells. Two classes of viral factors impact these events and contribute to virulence of per os infection without affecting virulence of BV.

The so-called pif (per os infectivity factor) genes of AcMNPV and their homologues are representative of the first class of factors. The pif genes are essential for establishing midgut infection and are highly conserved among all sequenced baculoviruses. Moreover, their absence is inconsequential to BV infectivity. AcMNPV p74, the founding member of this class, was described over a decade ago (Kuzio et al., 1989). Two more genes were identified subsequently in Spodoptera littoralis NPV and S. exigua NPV; these were SlNPV ORF 7 (pif) and SeNPV ORF 35 (pif-2), homologues of Ac119 and Ac022, respectively (Kikhno et al., 2002; Pijlman et al., 2003). AcMNPV p74 and pif encode ODV structural proteins and AcMNPV P74 is involved in the specific binding of ODV to midgut cells (Haas-Stapleton et al., 2004). The functions of PIF and PIF-2 are still unknown.

Members of the second class of factors promote rapid transmission of BV to tracheolar cells and thereby enhance virulence of infection initiated per os. This class of factors is diverse and includes PE38 (Milks et al., 2003) and GP64 expressed early, prior to virus replication (Washburn et al., 2003; Zhang et al., 2004). Such factors are of interest because, whilst not essential for in vivo infection, they ‘fine-tune’ virulence in host insects and their effects may vary among susceptible species.

Recently, Lapointe et al. (2004) reported that two members of the ‘11K gene family’, Ac145 and Ac150, enhance virulence of AcMNPV occlusions without affecting BV infectivity. The ‘11K genes' are predicted to encode small proteins of 90–110 aa that contain hydrophobic N termini and single copies of the so-called ‘C6 motif’ or ‘peritrophin-A domain’, thought to bind chitin (Dall et al., 2001; Tellam et al., 1999). The C6 motif also occurs within proteins encoded by diverse species within the ecdysozoan clade. Such proteins include various chitinases, mucins, peritrophins and other proteins incorporated within peritrophic membranes lining the guts of caterpillars and basal laminae of insect tracheae (Dall et al., 2001). Between the hydrophobic N terminus and the peritrophin-A domain, Ac150 also encodes a short stretch of basic and then acidic amino acids, with an RGD sequence separating the two. This is of note because RGD is an integrin-binding domain, and integrins make transmembrane connections to the cytoskeleton and may activate cellular signalling pathways (Hynes, 2002).

All baculovirus species infecting lepidopteran or hymenopteran larvae that have been sequenced to date contain one or more of the 11K homologues, and the apparent affinity of the proteins for chitin suggests a role during primary infection, possibly at the peritrophic-membrane interface. Lapointe et al. (2004), however, were unable to demonstrate chitin-binding activity for either Ac150 or Ac145, nor were they able to show that the absence of Ac150 alone had any adverse effect on virulence. The latter was a surprising result because deletion of Ac145 alone, or together with Ac150, reduced virulence in orally infected H. virescens larvae by 6- and 39-fold, respectively. Moreover, Ohkawa (1997) found that deletion of the Bombyx mori NPV homologue of Ac150, BmNPV ORF 126, reduced virulence in orally infected B. mori larvae. Our long-term interest in baculovirus pathogenesis in vivo led us to revisit the question of a possible role for Ac150 in oral infection. We generated an Ac150 deletion mutant, Ac{Delta}150, in which the hsp70/lacZ reporter cassette was inserted into the Ac150 ORF. In comparative bioassays with wild-type occlusions, we found that virulence of Ac{Delta}150 occlusions was decreased significantly in larvae of all three species tested (H. virescens, T. ni and S. exigua). Comparison of pathogenesis revealed that the only discernible role of Ac150 was to enhance establishment of primary midgut-cell infection, rather than to facilitate rapid transmission of BV. In this regard, Ac150 is in the same class as the pif genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid construction.
PCR was used to amplify the 5' and 3' regions of Ac150, using the AcMNPV EcoRI B fragment (which contains Ac150 and adjacent genes) as template. The 5' 502 bp region of Ac150 was amplified by using primer pairs 150A (5'-TAATAAAACTGGCCTGA-3') and 150B (5'-ACTGAACCCGTCGTCTG-3'), ensuring that Ac149 was left intact. The 3' 508 bp region of Ac150 was amplified by using primers 150C (5'-ATGGTACCAAATAAAATAAAATTTATATAGATTAATGAAATAAAATTTATATAGATT-3') and 150D (5'-ACTCGTAACCAGGATTC-3'), the former made longer by the addition of a KpnI site for use in determining fragment orientation within transfer plasmids (Fig. 1a) and also to make the annealing temperature similar to that of 150D. pBSKS+ (Stratagene) was cut with XbaI, then blunted by Klenow digestion and ligated to the 5' terminus of Ac150. The correctly oriented clone, designated p150-5'-BSKS, was digested with BamHI/XbaI and ligated to an hsp70/lacZ cassette (cleaved from pAcDZ1 by using the same enzymes; Zuidema et al., 1990) to form p150-5'-hsp70/lacZ-BSKS. This second plasmid was cut with SmaI and blunt-ligated to the 3' terminus of Ac150, generating the final transfer plasmid, p150-5'-hsp70/lacZ150-3'-BSKS, in which 60 % of the coding region of Ac150 was deleted (Fig. 1b). DNA sequencing of p150-5'-hsp70/lacZ150-3'-BSKS showed that the inserted sequence was orientated correctly (data not shown).



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Fig. 1. Construction and confirmation of Ac{Delta}150 and Ac{Delta}150R. (a) Schematic diagram of the AcMNPV ORF150 region in AcMNPV wild-type DNA. Flanking ORFs are indicated, as are the orientations and approximate locations of primers used to construct p150-5'-hsp70/lacZ150-3'-BSKS. (b) Schematic diagram of p150-5'-hsp70/lacZ150-3'-BSKS. The hsp70/lacZ cassette is flanked by the 5' and 3' termini of the Ac150 ORF. The orientations and approximate locations of primers used to confirm the genetic integrities of Ac{Delta}150 and Ac{Delta}150R are indicated. (c) PCR and agarose-gel electrophoresis analysis of Ac{Delta}150 and Ac{Delta}150R. PCR products of the expected size were generated when using 150E and lacZ 1 primers (lane 2) or 150F and lacZ 2 primers (lane 3) with Ac{Delta}150 DNA. No reaction products were generated when these primers were used with Ac{Delta}150R DNA (lanes 4 and 5). When primers 150E and 150F were used in conjunction with Ac{Delta}150R DNA, the PCR products generated were of the expected size (lane 6) and identical to those generated with wild-type E2 DNA (lane 7). The positions of molecular size markers (in kb) are indicated to the left of the blot.

 
Construction and genetic analysis of Ac{Delta}150 and Ac{Delta}150R.
An AcMNPV Ac150 deletion mutant was constructed by co-transfecting Spodoptera frugiperda (Sf-9) cells with p150-5'-hsp70/lacZ150-3'-BSKS and wild-type AcMNPV DNA. The deletion mutant, Ac{Delta}150, was identified by lacZ expression in infected Sf-9 cells and isolated after four rounds of plaque purification. Various restriction endonucleases were used to digest Ac{Delta}150 DNA prior to its analysis by 0·7 % agarose-gel electrophoresis and ethidium bromide staining; the restriction profiles indicated that the deletion mutant was constructed properly (data not shown). To further ensure the integrity of the DNA backbone of Ac{Delta}150, we constructed a revertant, Ac{Delta}150R, by co-transfecting Sf-9 cells with Ac{Delta}150 genomic DNA and a plasmid containing the AcMNPV EcoRI B fragment. Ac{Delta}150R, lacking lacZ expression, was isolated after three rounds of plaque purification. Ac{Delta}150R and wild-type AcMNPV DNAs were digested with various restriction endonucleases and compared by agarose-gel electrophoresis as above; the restriction profiles were indistinguishable (data not shown). The genetic integrities of Ac{Delta}150 and Ac{Delta}150R were further examined by PCR, with the products again analysed by agarose-gel electrophoresis and ethidium bromide staining (Fig. 1c). Primers 150E and 150F, designed to bind to the viral genomic sequences flanking Ac150 (Fig. 1b), were 5'-ATATCTTGTACTAGTGTCGGGGCGC-3' and 5'-CACAGTCGCCAGATTTGTTTGCCTCG-3', respectively. Primers lacZ 1 and 2, designed to bind to the 5' and 3' regions of hsp70/lacZ (Fig. 1b), were 5'-CACAATAACCAGTTTGTTTTGGGATTCTAG-3' and 5'-CAGTTGGTCTGGTGTCAAAAATAATAATAA-3', respectively. All sequencing data confirmed the correct genetic construction of Ac{Delta}150 and Ac{Delta}150R (data not shown).

Virus preparation.
Four viruses were used in the experiments described in this report: Ac{Delta}150 and Ac{Delta}150R (described above), AcMNPV-hsp70/lacZ (Engelhard et al., 1994) and AcMNPV E2, the parental wild-type virus (Smith & Summers, 1978). AcMNPV-hsp70/lacZ BV and ODV both have wild-type virulence levels in vivo (Engelhard et al., 1994; Washburn et al., 1995). Occlusion populations of each virus were generated from infected Sf-9 cells, harvested at 5 days post-infection and partially purified by sucrose-gradient centrifugation (Summers & Smith, 1987). Occlusions were suspended in a neutrally buoyant solution of glycerine and water (3 : 2, v/v) and quantified by using a haemocytometer (Washburn et al., 1995). ODV used in bioassays was liberated from occlusions by exposure to dilute alkaline saline and neutralized with 1 M Tris buffer. Undissolved occlusions and empty calyxes were removed by pelleting at 2000 g for 10 min; subsequently, ODV in the supernatant was banded by density-equilibrium centrifugation on continuous 25–59 % sucrose gradients for 1 h at 90 000 g. The resulting ODV bands were harvested and pooled, diluted 1 : 3 in PBS and pelleted at 90 000 g for 30 min. ODV pellets were collected in a minimal volume of PBS and aliquots of the two ODVs were quantified by using a BSA protein assay (Pierce) (Haas-Stapleton et al., 2004). To stabilize ODV, we added BSA to a final concentration of 10 µg ml–1 and dispensed small aliquots, which were stored at –20 °C until use. For bioassays, ODV inocula were thawed and diluted to the appropriate concentration in PBS immediately before use. BV was harvested at 72 h post-infection from the supernatant of Sf-9 cells infected with each of the viruses and titrated by immunoplaque assay on Sf-9 cells (Volkman & Goldsmith, 1982). For bioassays, BV stocks were diluted to the appropriate concentration with PBS and BSA (10 µg ml–1, final concentration). Stocks of all viruses were stored at 4 °C in the dark until use.

ODV content and nucleocapsid-packaging characteristics.
To compare ODV content of occlusions and nucleocapsid-packaging characteristics of Ac{Delta}150, Ac150{Delta}R and AcMNPV wild-type, ODV was harvested from 1·8x109 occlusions of each virus and equal volumes were subjected to density-equilibrium centrifugation as described above. The banding patterns of each virus were compared by visual inspection and photographed prior to fractionation with an ISCO density-gradient fractionator (model 640); A254 was measured and the relative areas beneath the peaks were calculated.

Insects and virus inoculation.
For all experiments, we used fourth-instar larvae of H. virescens, T. ni or S. exigua reared from eggs provided by the USDA Western Cotton Research Laboratory, Phoenix, AZ, USA. All larvae were reared in groups at 28±2 °C on a modified wheatgerm diet (Stoneville) until the onset of quiescence at the end of the third instar, indicative that larvae are preparing to moult to the fourth instar. For some experiments, large numbers of quiescent third instars were held between 4 and 15 °C until sufficient insects of the same developmental stage were available for testing (Washburn et al., 1995). Each larva was inoculated individually with occlusions, ODV or BV in 0·5–1·5 µl aliquots, using a microapplicator (Burkhard) fitted with a blunt- or sharp-tipped 32-gauge needle (for oral and intrahaemocoelic inoculations, respectively) mounted on a 1 ml tuberculin syringe [for details, see Washburn et al. (1995)]. For one experiment, suspensions of Ac{Delta}150 and AcMNPV wild-type occlusions additionally contained 1 % M2R dissolved in DMSO or just DMSO for control inocula (see Washburn et al., 1998).

Occlusions and ODV were administered orally by inserting the blunt-tipped needle through the mouth until the tip was well within the midgut lumen. BV was injected into the haemocoel by inserting the sharp-tipped needle through the planta of one of the prolegs, as described previously (Washburn et al., 1995). Larvae were inoculated orally within 15 min after moulting to the fourth instar (i.e. newly moulted larvae or 40) or 16 h after the moult (416). For all BV inoculations, we used fourth-instar larvae 24±6 h post-moult. After inoculation, test larvae were maintained individually in 25 ml plastic cups containing diet ad libitum in a growth chamber at 28±2 °C.

Bioassays and time-course experiments.
Bioassays were performed to compare the virulence of Ac{Delta}150 and Ac{Delta}150R occlusions, ODV and BV relative to those of AcMNPV wild-type in H. virescens, T. ni and S. exigua. For these and all additional assays described below, individual larvae were inoculated with varying dosages of inoculum (n=22–32 larvae per dosage) administered orally or intrahaemocoelically as described above. All larvae were maintained until pupation or death from polyhedrosis disease, and baculovirus-induced mortality was confirmed by microscopic examination (400x) of cadaver tissues for occlusions. For each of the three species, we established the oral dose–mortality relationships for Ac{Delta}150 by inoculating 40 larvae with various occlusion numbers. The dose–mortality relationship for each species was quantified by the method of least squares and regression equations were used to calculate the LD50 for each species. These values were compared with the LD50 of 40 larvae inoculated with wild-type AcMNPV occlusions. A minimum of five assays was used to calculate the wild-type LD50 for each species.

M2R is a stilbene-derived optical brightener known to bind chitin and damage the peritrophic membrane (Wang & Granados, 2000). To determine whether M2R affected the virulence of Ac{Delta}150, 40 and 416 T. ni were inoculated orally with 50 and 10 occlusions of Ac{Delta}150 or AcMNPV wild-type virus, respectively, in the presence or absence of 1 % M2R. These dosages were predicted to generate final mortalities of between 30 and 50 %, levels sufficiently low to quantify M2R mortality enhancement, if present, for both developmental cohorts. Additional bioassays were conducted to compare the virulence of Ac{Delta}150 and AcMNPV wild-type ODV in H. virescens and T. ni. In these experiments, identical dosages of between 0·1 and 100 pg of either Ac{Delta}150 or wild-type ODV were administered orally to larval cohorts of each species.

To evaluate the effects of deleting Ac150 on pathogenesis in vivo, we conducted a time-course experiment using 40 S. exigua inoculated with occlusions of either Ac{Delta}150 or AcMNPV-hsp70/lacZ. In this experiment, we used a dosage for each virus (determined from bioassays described above) that yielded final mortalities of ~85 %. At 4 h intervals during the first 24 h post-inoculation (p.i.), cohorts of between 26 and 32 larvae from each viral treatment were dissected and their midguts and associated tissues were removed. These tissues were processed to elucidate the blue {beta}-galactosidase reporter signal 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, 2003). For each host species, an additional cohort of 32 insects was inoculated orally with Ac{Delta}150 or AcMNPV-hsp70/lacZ to confirm that the dosages used yielded the same final mortality.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bioassays
In bioassays, we found that the virulence of Ac{Delta}150 BV was identical to that of wild-type AcMNPV BV following intrahaemocoelic injection into fourth-instar H. virescens, T. ni and S. exigua. In all three hosts, a dosage of 1 p.f.u. resulted in final larval mortalities of between 52 and 86 %, depending on the host species (Table 1). Similarly, oral administration of 10 occlusions of either Ac{Delta}150R or AcMNPV wild-type virus to 40 larvae yielded comparable levels of mortality in each of the hosts (Table 2), providing biological evidence that the genetic backbone of Ac{Delta}150 was wild-type. In sharp contrast, significant differences in the virulence of occlusions of the Ac150 deletion mutant compared with wild-type virus were observed in all three host species (Fig. 2). For each species, significantly more occlusions were required per LD50 for Ac{Delta}150 compared with wild-type virus, but the amount varied among hosts. For 40-inoculated S. exigua and H. virescens, the Ac{Delta}150 dosages required were 4·1- and 5·6-fold greater than for wild-type, respectively, and for T. ni, the dosage was 18-fold greater (Fig. 2). These results indicated that Ac150 indeed had an enhancing effect on per os infection and that the effect was host-specific.


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Table 1. Mortalities (%) of H. virescens, T. ni and S. exigua inoculated intrahaemocoelically as feeding fourth instars with 1 p.f.u. AcMNPV E2 or Ac{Delta}150

Each cohort contained 32 larvae.

 

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Table 2. Mortalities (%) of H. virescens, T. ni and S. exigua inoculated orally as newly moulted fourth instars with 10 occlusions of either AcMNPV E2 or Ac{Delta}150R

Each cohort contained 20–32 larvae.

 


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Fig. 2. Dose–response curves for S. exigua (a), H. virescens (b) and T. ni (c) inoculated orally as newly moulted fourth instars with occlusions of Ac{Delta}150. The open circles indicate the LD50 value of AcMNPV E2, the wild-type parental strain for Ac{Delta}150. Each point represents the proportion of larvae dying from polyhedrosis disease in a cohort of 22–32 insects; the LD50 values for AcMNPV E2 represent the mean of a minimum of five assays, each using 26–32 insects. Regression lines for Ac{Delta}150 were fitted by the method of least squares, and numbers adjacent to the dotted line (50 % mortality) beside each regression line represent Ac{Delta}150 LD50 values. Regression equations for Ac{Delta}150 are as follows: S. exigua, y=0·71x+26·8, r2=0·96; H. virescens, y=0·36x+29·7, r2=0·88; T. ni, y=0·18x+17·6, r2=0·82.

 
Time-course experiments
In order to determine how the loss of Ac150 impacted on virulence of orally inoculated AcMNPV, we conducted a time-course experiment in which we used the lacZ reporter signal as a marker of infection to compare early viral pathogenesis of Ac{Delta}150 with AcMNPV-hsp70/lacZ in 40-inoculated S. exigua. In doing so, we defined several critical benchmarks of infection for each virus including: (i) the temporal onset of primary infection of midgut cells; (ii) the rate of primary infection of midgut cells; (iii) the number of infection foci generated per occlusion; (iv) the number of foci required to achieve comparable mortality; and (v) the rate at which primary cellular targets transmitted BV to secondary targets within the tracheal epidermis. In S. exigua inoculated with either Ac{Delta}150 or AcMNPV-hsp70/lacZ, lacZ expression (indicative of infection) was first observed at 8 h p.i. (Fig. 3). For both viral treatments, the proportion of LacZ-positive larvae increased rapidly between 8 and 16 h p.i., and by 16 h p.i. the proportions of larvae expressing LacZ were roughly equivalent to the final mortality determined in the companion Ac{Delta}150 and AcMNPV-hsp70/lacZ bioassays (~85 %) (Fig. 3a). Thus, the rate and timing of primary infection by Ac{Delta}150 were the same as for the control virus. Similarly, we detected no significant differences in either the numbers of foci at any time point during the first 24 h p.i. (analysis not shown) or in the number of foci required to generate the same final mortality (Fig. 3b). Notably, however, five times as many occlusions of Ac{Delta}150 were administered as AcMNPV-hsp70/lacZ, suggesting that control ODV was 5-fold more efficient at establishing primary foci than the deletion mutant. As with primary infection, the onset and rate of secondary infection of the tracheal epidermis of host larvae were identical for both viruses. Tracheal cells infected by the ODVs of Ac{Delta}150 and AcMNPV-hsp70/lacZ were first observed at low frequencies at 8 h p.i. and increased linearly with identical slopes until 24 h p.i., when sampling was curtailed (Fig. 3c). As a consequence, cohorts of S. exigua larvae became systemically and fatally infected at exactly the same rate by the mutant and control viruses (Fig. 3d). Thus, the only significant difference between early viral pathogenesis of Ac{Delta}150 and AcMNPV-hsp70/lacZ in S. exigua larvae was in the number of primary foci generated by each occlusion. Finally, in a second set of time-course experiments, we quantified the mean time to death for H. virescens larvae challenged orally or intrahaemocoelically with occlusions or BV, respectively, of Ac{Delta}150 or AcMNPV wild-type, using dosages that yielded a final mortality of ~85 % for each treatment. By both routes of introduction, the mean time to death by polyhedrosis disease (~100 h) was statistically the same for cohorts challenged with either Ac{Delta}150 or AcMNPV E2 (data not shown).



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Fig. 3. Pathogenesis of Ac{Delta}150 and AcMNPV-hsp70/lacZ in S. exigua following oral inoculation of newly moulted fourth-instar larvae with occlusions of either Ac{Delta}150 (LD84) or AcMNPV-hsp70/lacZ (LD83). Each point represents data from a cohort of 23–26 larvae. (a) Percentage of larvae with LacZ expression in tissues at various h p.i. (b) Mean number of foci per LacZ-positive larva; error bars represent ±1SEM. (c) Percentage of viral foci containing one or more LacZ-positive tracheal epidermal cell. Regression lines were determined by the method of least squares: for Ac{Delta}150 (solid line), y=4·2x–10, r2=0·95; for AcMNPV-hsp70/lacZ (dashed line), y=4·6x–22, r2=0·98. (d) Percentage of larvae containing one or more LacZ-positive tracheal epidermal cell. Regression lines were determined by the method of least squares: for Ac{Delta}150 (solid line), y=4·5x–19·2, r2=0·89; for AcMNPV-hsp70/lacZ (dashed line), y=4·5x–13·2, r2=0·91.

 
ODV content and nucleocapsid-packaging characteristics among Ac{Delta}150, Ac{Delta}150R and AcMNPV E2
In order to determine whether there were differences in ODV content per occlusion or in nucleocapsid distribution among ODV populations that could account for differences in virulence, we banded ODV isolated from equal numbers of occlusions of Ac{Delta}150, Ac{Delta}150R and wild-type AcMNPV on matching sucrose-density gradients. In this experiment, no obvious differences in ODV concentration or in banding patterns were observed (Fig. 4a). Further analysis by density-gradient fractionation and A254 measurement also failed to reveal any differences either in the amounts of ODV or in the relative proportion of each ODV band (Fig. 4b). Thus, the virion populations within occlusions of both the deletion mutant and its revertant were the same as wild-type virus with regard to ODV concentration and nucleocapsid distribution.



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Fig. 4. Sucrose-gradient separation of ODV from similar occlusion numbers (~2x109) of Ac{Delta}150, AcMNPV E2 and Ac{Delta}150R. (a) ODV bands in the sucrose gradient after centrifugation. (b) Relative frequencies (%) of ODV bands from (a) following analysis using an ISCO density-gradient fractionator (model 640) and an absorbance monitor (model UA-5) at a wavelength of 254 nm. Each ODV band was quantified by the area under each peak.

 
Effects of M2R on virulence of Ac{Delta}150 and AcMNPV wild-type administered orally
Another possible explanation for the reduced number of primary foci by Ac{Delta}150 could be that Ac150 enhances ODV contact with primary target cells, possibly by facilitating virion passage across the peritrophic membrane. We conducted an experiment, therefore, to see whether we could increase Ac{Delta}150 oral virulence to wild-type levels by physically damaging the peritrophic membrane with M2R. M2R is a chemical with chitin-binding properties that has been shown to dissociate proteins from lepidopteran peritrophic membranes, presumably by competing with the chitin-binding domains of the proteins (Wang & Granados, 2000). We reasoned that if Ac150 acted in a manner similar to M2R, and if Ac{Delta}150 occlusions were administered at fivefold the dosage of wild-type occlusions, then M2R should enhance infectivity of the Ac{Delta}150 occlusions to a greater degree than the wild-type. We orally inoculated 40 and 416 larvae of T. ni (the host exhibiting the greatest resistance to fatal infection in the absence of Ac150) with 50 occlusions of Ac{Delta}150 or 10 occlusions of AcMNPV wild-type in the presence or absence of 1 % M2R (Washburn et al., 1998). M2R addition to the inoculum yielded no significant change in mortality in the 40 cohorts challenged with either virus (Fig. 5). In the 416 cohorts, T. ni larvae exhibited similar levels of developmental resistance to both viruses in the absence of M2R, whilst in the presence of M2R, mortality levels were rescued to 40 levels. Thus, although M2R enhanced mortality significantly in the 416 cohorts, the level of enhancement was the same for mutant and wild-type, even though the mutant virus was present at fivefold the concentration (Fig. 5). Hence, in both T. ni developmental cohorts, disruption of the physical integrity of the peritrophic membrane by M2R failed to rescue the reduction in oral virulence when Ac150 was deleted, suggesting that the mode of action of Ac150 was different from that of M2R.



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Fig. 5. Mortality levels of T. ni inoculated orally with 10 occlusions of AcMNPV E2 or 50 occlusions of Ac{Delta}150 immediately after moulting to the fourth instar (40) or 16 h after the moult (416). The viral inocula used for the M2R treatment contained 1 % M2R. Both control and M2R inocula were delivered in 1 µl dosages. Each histogram bar represents the mean of three replicates (±1SEM), each using 26–32 insects.

 
Comparison of virulence of Ac{Delta}150 and AcMNPV wild-type ODV administered orally
Lapointe et al. (2004) reported that Ac150 was associated with occluded and pre-occluded virus late and very late during infection. To determine whether Ac150 activity tracked with the ODV particle, we isolated the ODV from wild-type and Ac{Delta}150 occlusions and compared their virulence in 40 H. virescens and T. ni. In both host species, there were no significant differences in the dose–mortality relationships of the deletion mutant and wild-type virus (Fig. 6). Thus, whilst occlusions of Ac{Delta}150 were 5·6- and 18-fold less virulent than occlusions of wild-type virus in H. virescens and T. ni, respectively, the purified ODVs of both exhibited identical virulence following oral administration.



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Fig. 6. Dose–response curves for H. virescens (a) and T. ni (b) inoculated orally as newly moulted fourth instars with varying dosages of AcMNPV E2 or Ac{Delta}150 ODV. Each point represents a cohort of 26–32 insects and regression equations were determined by the method of least squares. The equations for Ac{Delta}150 (solid line) and AcMNPV E2 (dashed line) in H. virescens are: Ac{Delta}150, y=24·3logx+60·8, r2=0·76; AcMNPV E2, y=23·2logx+58·9, r2=0·79. The equations for Ac{Delta}150 (solid line) and AcMNPV E2 (dashed line) in T. ni are: Ac{Delta}150, y=20·2logx+23·9, r2=0·98; AcMNPV E2, y=23·8logx+58·9, r2=0·79.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our results showed unequivocally that Ac150 enhanced oral infection; from 4- to 18-fold more Ac{Delta}150 occlusions than wild-type occlusions were required to achieve 50 % mortality, depending on the host species. No differences were found in ODV content per occlusion, nor in nucleocapsid distribution among the ODV populations, that could explain the observed differences in virulence. In time-course experiments using lacZ expression to elucidate the events of early pathogenesis, we found that the only effect of the absence of Ac150 during infection was to reduce the number of foci generated by each occlusion. Hence, Ac150 affected virulence of per os infection by increasing the efficiency of establishing primary focus formation. No differences were detected either in time to death following oral infection, or in virulence following intrahaemocoelic injection of BV into any of the hosts. Ac150, therefore, can be considered a per os infection factor that mediates, but is not essential for, oral infection.

Interestingly, whilst the occlusions of Ac{Delta}150 were less infectious orally than wild-type occlusions, the isolated ODVs had the same infectivity. These results suggested that exposure to dilute alkaline saline inactivated Ac150 or that it was lost during ODV purification, or both. The lack of Ac150 activity associated with purified ODV is consistent with the findings of Braunagel et al. (2003), who found no evidence of Ac150 in AcMNPV ODV by using multiple analytical approaches.

To test whether Ac150 facilitated passage of ODV across the peritrophic membrane (which could explain the reduced efficiency of primary infection), we inoculated Ac{Delta}150 occlusions in the presence of the stilbene-derived optical brightener, M2R, which is known to release proteins from the peritrophic membrane and cause holes to form (Wang & Granados, 2000). In our experiments, addition of 1 % M2R failed to enhance mortality levels generated by the deletion mutant to expected levels if Ac150 worked by a similar mechanism to M2R. This result was consistent with the lack of chitin-binding activity reported by Lapointe et al. (2004).

It is possible that Ac150 has a role in signalling. Integrins are known to propagate signalling when bound by a ligand, and Ac150 has an RGD integrin-binding motif in the middle of a cluster of charged amino acids. Alternatively, microvilli of midgut cells are coated heavily with glycosylated proteins and the peritrophin-A domain of Ac150 may bind one of these. A number of membrane-bound receptors for growth factors and cytokines are glycosylated, and evidence has indicated that oligosaccharide moieties are crucial for the functions of some of those receptors (Takahashi et al., 2004). Whether or not Ac150 binds to midgut cells at all, however, remains to be determined.


   ACKNOWLEDGEMENTS
 
Financial support was provided the Torrey Mesa Research Institute and Syngenta Research and Technology, USDA NRICG #2001-35302-10883 and USDA NRICG #2002-35302-12601. We are grateful to the USDA Western Cotton Research Laboratory, Phoenix, AZ, for providing eggs of S. exigua, T. ni and H. virescens for use in this study. We thank Annette R. Rowe, Kamal Gandhi, Ronika Sitapara and Eric Sid for technical assistance with the experiments. We also thank Andrew O. Jackson for use of his density-gradient fractionator.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 28 January 2005; accepted 14 March 2005.