1 Department of Plant and Microbial Biology, 251 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA
2 DuPont Agricultural Products, Stine-Haskell Research Center, PO Box 30, Elkton Road, Newark, DE 19714, USA
Correspondence
Jan Washburn
janwash{at}nature.berkeley.edu
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ABSTRACT |
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Published ahead of print on 15 November 2002 as DOI 10·1099/vir.0·18701-0.
Present address: Pioneer Hi-Bred International, 7250 NW 62nd St., Johnston, IA 50131, USA.
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Introduction |
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Historically, species within the genus Nucleopolyhedrovirus have been designated as either M or S, referring to whether the ODV particles contain multiple (M) or single (S) nucleocapsids. In contrast, the BV of both SNPVs and MNPVs are packaged singly. The biological consequence of the M phenotype is that the midgut cells of the host are infected with multiple nucleocapsids contained within the ODV envelope. There are many more species of MNPV than SNPV described (Vail et al., 1999; Volkman, 1997
), and all known MNPVs infect species in the Lepidoptera, the most recently derived insect order. In addition to lepidopterans, however, SNPVs have been isolated also from species in the orders Hymenoptera and Diptera. These host affinities suggest that the progenitor Nucleopolyhedrovirus was probably an SNPV (Rohrmann, 1986
) and indicate that the M phenotype is a more recently acquired baculovirus trait. While very little is known about what determines the number of nucleocapsids packaged per virion for the MNPVs, the evolutionary history and host ranges of the NPVs suggest that the M phenotype evolved from the S phenotype and therefore, may incur a selective advantage.
To determine if there is a functional significance between these two ODV phenotypes during the early stages of infection, we compared virus pathogenesis and infection kinetics of an MNPV, Autographa californica MNPV (AcMNPV), and an SNPV, Helicoverpa zea SNPV (HzSNPV), in larvae of Heliothis virescens (Lepidoptera: Noctuidae). AcMNPV is the type species of the Nucleopolyhedrovirus genus (Blissard et al., 2000) and can infect at least 32 lepidopteran species within 12 families (Granados & Williams, 1986
); this broad host range shows that AcMNPV possesses an effective strategy for fatally infecting larval lepidopterans. In contrast, HzSNPV is reported to have a relatively narrow host range, encompassing species in the Heliothinae, a subfamily within the Noctuidae which includes H. virescens (Allen & Ignoffo, 1969
; Getting & McCarthy, 1982
; Granados & Williams, 1986
; King & Coleman, 1989
; Mitter et al., 1993
; Black et al., 1997
). HzSNPV also was the first baculovirus pesticide to be introduced into the commercial marketplace for control of lepidopteran pests (Black et al., 1997
), and although commercialization failed, basic research has shown that HzSNPV pathogenesis is similar to that of AcMNPV in noctuid larvae (Granados, 1978
; Granados & Williams, 1986
; Washburn et al., 2001
).
To compare virus pathogenesis in vivo, we used recombinants of AcMNPV and HzSNPV containing identical hsp70/lacZ reporter-gene cassettes and a host insect fully permissive for both NPVs, newly moulted fourth instar larvae of H. virescens. With dosages that yielded similar levels of larval mortality, we conducted time-course experiments comparing the strategies of the two pathogens for establishing primary and secondary infections in vivo. We also examined their BV infection kinetics in a primary culture of H. virescens haemocytes. Our findings revealed that H. virescens haemocytes infected in vitro by AcMNPV-hsp70/lacZ or HzSNPV-hsp70/lacZ have similar infection kinetics. In orally infected H. virescens larvae, the kinetics of primary and secondary infection were also similar, although the onset by AcMNPV-hsp70/lacZ in midgut cells lagged behind HzSNPV-hsp70/lacZ by 6 h. Notably, secondary infection of tracheolar cells was initiated at the same time, 12 h post-inoculation (p.i.), suggesting a midgut transit time of 2 h for AcMNPV and 8 h for HzSNPV. The infection strategies of both NPVs resulted in the timely movement of BV into the tracheal epidermis, countering the host's response of avoiding mortal infection by sloughing infected midgut cells.
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Methods |
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Virus preparation and quantification.
AcMNPV-hsp70/lacZ (E2 parental strain) (Engelhard et al., 1994) and HzSNPV-hsp70/lacZ (Elcar HzSNPV-1 parental strain) (Washburn et al., 2001
) were used for the experiments described herein. These constructs contain all of the wild-type viral genes from their respective parental strains and the Esherichia coli
-galactosidase gene driven by the Drosophila hsp70 promoter. In cultured insect cells infected with either AcMNPV-hsp70/lacZ or HzSNPV-hsp70/lacZ, lacZ is expressed along with early viral genes, permitting us to monitor infection in host tissues by assaying for the presence of the enzyme. Occlusions of AcMNPV-hsp70/lacZ and HzSNPV-hsp70/lacZ were isolated from liquefied cadavers of H. virescens and partially purified by centrifugation on sucrose gradients (Summers & Smith, 1987
). Subsequently, occlusions were pelleted and resuspended in water, then diluted in a neutrally buoyant glycerin/water solution (3 : 2, v/v) (Washburn et al., 1995
). In bioassays with H. virescens larvae, both recombinants produced mortalities equivalent to their parental wide-type strains when administered orally with the same number of occlusions (data not shown). The occlusion dilutions used in this study were stored at 4 °C in the dark and quantified using a haemocytometer (minimum of five counts for each inoculum).
Comparative replication kinetics.
Because there are no available cell lines that can be infected by both AcMNPV and HzSNPV, we used a primary cell culture of haemocytes from larval H. virescens to compare virus replication kinetics in vitro. Haemocytes were isolated according to Pech et al. (1994) with some modifications. All centrifugation steps were performed at 4 °C. Haemocytes were pelleted by centrifugation in an Eppendorf 5417 C centrifuge at 334 g for 8 min. After incubation in anticoagulant buffer (0·098 M NaOH, 0·186 M NaCl, 0·017 M EDTA and 0·041 M citric acid, pH 4·5), haemocytes were pelleted (260 g for 3 min) and washed twice in Excell-400 medium (JRH Sciences) by centrifugation. The final pellet was resuspended in 50 µl Excell-400 medium and cell concentrations were estimated with a haemocytometer. To quantify BV production by haemocytes, 5x103 cells were placed in triplicate wells of four replica plates already containing 70 µl TC100 medium plus 20 % FBS and AcMNPV-hsp70/lacZ or HzSNPV-hsp70/lacZ at an m.o.i. of 10 p.f.u. per cell. Cells were infected for 2 h at room temperature, and viral inocula removed and replaced with ice-cold medium. Cells were rinsed twice more by centrifugation (200 g for 3 min) in a Beckman GPR tabletop centrifuge. After the final centrifugation step, we added 80 µl of medium to each well; we then removed 10 µl of medium from each well to determine the amount of residual inoculum. Haemocyte cultures were maintained at 28 °C and at 12, 24, 48 and 72 h p.i., 40 µl of medium were removed from three wells of a replica plate and transferred to individual 500 µl microfuge tubes. Tubes were held at 4 °C until all samples were collected, and plaque assays were performed with the appropriate cell line: Sf9 cells for AcMNPV-hsp70/lacZ (Engelhard et al., 1994
) and AM1 cells for HzSNPV-hsp70/lacZ (McIntosh et al., 1981
). Significantly, we determined that H. virescens haemocytes gave the same results in plaque assays of AcMNPV-hsp70/lacZ as did Sf9 cells, and also HzSNPV-hsp70/lacZ as did AM1 cells (D. Trudeau, unpublished data).
Mini time-course experiments were conducted to determine the onset of lacZ expression in vitro. Primary haemocyte cultures were infected at an m.o.i. of 10 and sampled at 1 h intervals for 5 h; samples were processed for lacZ expression as described previously (Engelhard et al., 1994) and examined for blue signals of expression.
Determination of ODV per occlusion.
The numbers of ODV per occlusion of AcMNPV-hsp70/lacZ and HzSNPV-hsp70/lacZ were quantified by transmission electron microscopy (TEM). Occlusions of both recombinants were prepared for TEM following standard protocols. To assess the number of ODV per occlusion, for each virus we printed photographs of sections (15 000x) on standardized paper and cut out the images from 100 different occlusions. The paper image of each occlusion cross section was then weighed, and the number of visible ODV was recorded. Weight provided an accurate measure of occlusion cross-sectional area (linear least-squares regression: AcMNPV-hsp70/lacZ, r2=0·94; HzSNPV-hsp70/lacZ, r2=0·99) and therefore, allowed a correlation between occlusion cross-sectional area (wt) and number of ODV.
Bioassay and time-course experiments.
For oral bioassays and time-course experiments, occlusion suspensions in 1·0 µl aliquots were inoculated through the mouth into the lumen of the anterior midgut of newly moulted (i.e. within 15 min of shedding the third instar cuticle) fourth instar H. virescens using a syringe fitted with a 32 gauge blunt-tip needle mounted on a microapplicator (Burkard Scientific). After inoculation, larvae were maintained in a growth chamber at 28±2 °C under constant illumination in individual 25 ml plastic cups containing diet ad libitum. To determine dosages for time-course experiments, we conducted bioassays in which cohorts of 32 or more larvae were inoculated as described above using varying occlusion numbers of AcMNPV-hsp70/lacZ or HzSNPV-hsp70/lacZ and maintained until death or pupation. Dosages of 15 and 20 occlusions of HzSNPV-hsp70/lacZ and AcMNPV-hsp70/lacZ, respectively, were selected for the experiments described here; in four replicated bioassays, 15 occlusions of HzSNPV-hsp70/lacZ produced an average mortality of 84 % (range=7594 %), and 20 occlusions of AcMNPV-hsp70/lacZ yielded an average larval mortality of 87 % (range=8688 %). To assess larval susceptibility to systemic infection, we injected BV of the recombinants directly into the haemocoel of fourth instar larvae using a 32 gauge sharp-tipped needle, as described previously (Engelhard et al., 1994; Engelhard & Volkman, 1995
; Washburn et al., 1995
).
For time-course experiments, we inoculated large numbers of newly moulted fourth instar H. virescens larvae with AcMNPV-hsp70/lacZ or HzSNPV-hsp70/lacZ. At various times during the first 24 h p.i., cohorts consisting of 2633 larvae were sacrificed, and their midgut and associated tissues were removed and assessed for lacZ expression (Engelhard et al., 1994; Washburn et al., 1995
). These tissue preparations were examined for lacZ expression with stereo (1050x) and/or compound microscopy (100480x) to quantify foci of infection and to identify infected cell types (Washburn et al., 1995
, 2001
). For each time-course experiment, an additional cohort of 32 insects was retained as an internal control bioassay to confirm mortality levels. We conducted several time-course experiments with each of the recombinants, and the results were the same among experiments. The data used for the analyses of virus pathogenesis presented here were from experiments in which the tissues of 278 and 266 H. virescens challenged with AcMNPV-hsp70/lacZ and HzSNPV-hsp70/lacZ, respectively, were examined for lacZ expression.
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Results |
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In order to compare the kinetics of larval infection by AcMNPV- hsp70/lacZ and HzSNPV-hsp70/lacZ, we used least-squares regression to characterize the temporal patterns of lacZ expression in cohorts of H. virescens. We found that for each recombinant, the temporal increase in the proportion of LacZ-positive larvae during the first 24 h p.i. was well described by a simple linear equation (Fig. 3). While there was a 6 h difference in the onset of midgut lacZ expression in larvae inoculated with AcMNPV-hsp70/lacZ relative to those inoculated with HzSNPV-hsp70/lacZ, the similarity in slope values (ratio of 1·1 : 1) indicates that (after adjusting for the 6 h difference in initial onset) initiation of infection of H. virescens larvae by both viruses proceeded at comparable rates.
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Discussion |
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Compared to AcMNPV-hsp70/lacZ, fewer HzSNPV-hsp70/lacZ occlusions were required to achieve the same mortality level in H. virescens larvae, and those fewer occlusions generated more than twice the number of primary foci. This result indicated that on a per occlusion basis (the natural unit of infection), the SNPV was the more efficient of the two, but on a per virion basis, the MNPV was more efficient. Previously, we showed that AcMNPV-hsp70/lacZ ODV particles containing multiple nucleocapsids were more efficient than AcMNPV-hsp70/lacZ ODV containing a single nucleocapsid in establishing mortal infection in orally inoculated T. ni (Washburn et al., 1999). The higher numbers of foci of HzSNPV-hsp70/lacZ were correlated with more virions per occlusion relative to AcMNPV-hsp70/lacZ (Fig. 5
), but other factors such as a greater efficiency of establishing and/or maintaining primary infections by the SNPV may also modulate primary numbers of foci.
In our time-course experiment, despite the 6 h difference in the onset of primary infections, the onset and rate for secondary infection of tracheolar cells were virtually identical (Fig. 7). We observed an 8 h delay between midgut and tracheal infection by HzSNPV-hsp70/lacZ compared to only a 2 h delay during AcMNPV-hsp70/lacZ pathogenesis. By any study published to date, 2 h is much too short a time for complete replication of a baculovirus. Thus, these results are consistent with the hypothesis that the M character of AcMNPV ODV permits repackaging of parental nucleocapsids as BV, facilitating early infection of the tracheal epidermis before de novo virus replication within infected midgut cells (Washburn et al., 1999
, 2002
). Repackaging the nucleocapsids of AcMNPV ODV is made possible by early expression of GP64, a BV envelope protein absent from ODV (Keddie & Volkman, 1985
; Volkman, 1986
; Blissard & Rohrmann, 1989
; Jarvis & Garcia, 1994
) but essential for establishment of system infections in T. ni and H. virescens infected per os (Monsma et al., 1996
; J. O. Washburn, unpublished data). GP64 is widely conserved among the group I MNPVs (reviewed by Garrity et al., 1997
) and is unusual among viral proteins in that its synthesis occurs hours before other structural proteins are made. The M character, coupled with early GP64 expression, provides a mechanism whereby AcMNPV BV can be transmitted to tracheal cells before virus replication is complete. In contrast, HzSNPV lacks both GP64 (Chen et al., 2002
) and the M character; thus, BV transmission must be delayed until virus replication occurs in the primary target.
By 24 h p.i., the proportion of LacZ-positive H. virescens larvae challenged with HzSNPV-hsp70/lacZ (83 %) was equivalent to the final larval mortality (84 %) and notably, all infected larvae in this cohort supported one or more systemic foci of infection. In contrast, only 57 % of larvae inoculated with AcMNPV-hsp70/lacZ were infected at 24 h p.i., a level significantly below the final mortality of 87 %; moreover, in a significant number of these larvae, secondary infections had not been established yet (Fig. 2). Our regression equations describing AcMNPV-hsp70/lacZ primary and secondary infection kinetics (Figs 3 and 8
) predicted that infection levels should equal the final mortality at 30·4 and 37·4 h p.i., respectively, times that coincide with the onset of the first moult after inoculation. These findings suggest that functional primary infections were established by AcMNPV-hsp70/lacZ throughout the fourth instar (Washburn et al., 1995
). At 32 h p.i., 20 % of the H. virescens cohort inoculated with AcMNPV-hsp70/lacZ were quiescent and non-feeding, indicating that they were preparing to moult. At this stage, larvae void the contents of the gut lumen and shed infected midgut cells, thereby eliminating both residual ODV and primary infections (Washburn et al., 1995
); thus, levels of secondary infection at this premoult stage are predictive of final mortality levels (Engelhard & Volkman, 1995
; Washburn et al., 1995
). The fact that our equations predicted the final mortality level coincident with the premoult stage suggests that our linear models based on the LacZ signal accurately reflect the kinetics of AcMNPV-hsp70/lacZ.
The extreme sensitivity of H. virescens to BV entry of either NPV into the haemocoel underscores the importance for establishing even a single viral focus within the tracheal epidermis, because doing so ensures a productive infection and death of the host. In order to escape virus death, larvae must slough ODV-infected midgut cells prior to their transmission of BV to the haemocoel. Our experiments showed that some primary cellular targets infected by AcMNPV-hsp70/lacZ and HzSNPV-hsp70/lacZ were shed from the midgut as early as 16 h p.i., many hours before the onset of moulting. Sloughing of AcMNPV-infected midgut cells has been observed previously and photographed (Fig. 2A) (Keddie et al., 1989
) and may be a widespread and effective host defence against midgut-initiated virus infection. In our study, we could identify sloughing only for primary foci that successfully transmitted BV to the tracheal epidermis (i.e. via lacZ expression restricted to the tracheal epidermis), and frequency data on these foci suggested that midgut cells infected by AcMNPV-hsp70/lacZ ODV were sloughed at higher rates than those infected by HzSNPV-hsp70/lacZ (Fig. 6
). For HzSNPV-hsp70/lacZ, foci numbers rose continuously for 16 h after the onset of primary infection (i.e. 420 h p.i.); thus, if infected midgut cells were sloughed prior to 16 h p.i., the rate of loss was insufficient to counter the rise in new primary infections. Moreover, by 16 h p.i., 89 % of the LacZ-positive larvae (65 % of the cohort; Fig. 2
) inoculated with HzSNPV-hsp70/lacZ already supported secondary tracheal foci, indicating that subsequent sloughing would not have cleared infections. Throughout primary infection, numbers of AcMNPV-hsp70/lacZ foci remained substantially below those achieved by HzSNPV-hsp70/lacZ and reached a maximum at 16 h p.i., only 6 h after the onset of primary infection. We showed previously that when H. virescens larvae were orally inoculated progressively later during the fourth instar, AcMNPV-hsp70/lacZ exhibited a concomitant decreasing ability to establish and/or maintain primary infection (Washburn et al., 1998
). The delay in the onset of AcMNPV-hsp70/lacZ primary infection, the decline in numbers of primary foci after 16 h p.i. and the higher frequency of foci lacking primary infections provide evidence that the cellular targets infected by the MNPV were sloughed more frequently than those infected by the SNPV. This effect was counteracted, however, by the shorter time-lag between primary and secondary AcMNPV-hsp70/lacZ infection.
In summary, we have shown that both AcMNPV-hsp70/lacZ and HzSNPV-hsp70/lacZ transiently infect midgut columnar cells, which subsequently transmit BV to the host's haemocoel by infecting tracheolar cells. H. virescens larvae sloughed primary cellular targets of both NPVs, and the two pathogens countered this host defence in different ways. HzSNPV-hsp70/lacZ initiated primary infections more quickly and in greater numbers than AcMNPV-hsp70/lacZ but secondary infection by the SNPV was delayed by 8 h from the onset of primary infection. In contrast, AcMNPV-hsp70/lacZ established secondary infection only 2 h after establishing primary infections and continued to establish primary infections late into the instar. The difference in time intervals between primary and secondary infections support the hypothesis that the earliest secondary targets of the SNPV were infected by newly synthesized BV, whereas those of the MNPV were infected by parental ODV nucleocapsids repackaged as BV. Remarkably, BV transmission by both viruses to secondary target cells occurred at the same rate and time, suggesting that the infection strategies of both HzSNPV and AcMNPV have been selected for rapid establishment of systemic infection. To test directly if the earliest tracheal infections by AcMNPV are established by repackaged ODV nucleocapsids, we plan to label nucleocapsids radioactively and monitor their distribution in larval tissues.
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ACKNOWLEDGEMENTS |
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Received 9 July 2002;
accepted 29 October 2002.