Infection of wild-type Autographa californica multicapsid nucleopolyhedrovirus induces in vivo apoptosis of Spodoptera litura larvae

Ping Zhang1, Kai Yang1, Xiaojiang Dai1, Yi Pang1 and Deming Su2

State Key Laboratory for Biocontrol and Institute of Entomology, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, People’s Republic of China1
Virology Research Unit, Fudan University, Shanghai 200433, People’s Republic of China2

Author for correspondence: Yi Pang. Fax +86 20 8403 7472. e-mail LS12{at}ZSU.EDU.CN


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Direct evidence of in vivo apoptosis of Spodoptera litura larvae was demonstrated by haemocoel inoculation with wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virus (BV). In sharp contrast to natural infection, cadavers did not melt, liquefy and melanize. Typical morphological changes of apoptosis in insect haemocytes post-infection, including blebbing of the cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, were observed by light and electron microscopy. Total DNAs extracted from virus-infected haemocytes showed DNA ladders. Cleavage of chromatin DNA by endogenous endonucleases were detected in the cells of most tissues cells, including epithelial cells and fat body cells, using terminal dUTP nick end labelling assays. Virogenic stroma and viral nucleocapsids could be seen in the nuclei of a few haemocytes. Yields of BV and OV (occluded virus) produced from the infected S. litura larvae were much lower than from the infected S. exigua larvae. These data suggest that host apoptotic responses to virus infection reduce AcMNPV spread at the level of the organism and that apoptosis could be a host-range limiting factor for baculovirus infections.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Apoptosis, or programmed cell death, is a controlled biochemical pathway distinguishable from cell necrosis by characteristics that include cellular shrinkage, membrane blebbing and chromatin condensation (Wyllie et al., 1980 ). It plays a vital role in embryonic development, tumourigenesis and maintenance of homeostasis in continuously renewing tissues by eliminating redundant, unwanted or damaged cells from organisms. Apoptosis can be induced by a diverse range of stimuli, including virus infection (Thompson, 1995 ). Many viruses, including baculoviruses (Clem, 1997 ), are known to regulate apoptosis of host cells during infection (Razvi & Welsh, 1995 ; Teodoro & Branton, 1997 ; O’Brien, 1998 ). In the insect cell system, in vitro replication of baculoviruses was reduced by apoptosis and in vivo infectivity reduction was hypothesized to correlate with apoptosis (Clem & Miller, 1993 ). However, no supporting data could demonstrate that in vivo apoptosis occurred post-infection (p.i.) (Clem & Miller, 1993 ). This group of viruses has evolved two families of anti-apoptotic genes, p35 and iap, to suppress this host defence system. These two genes use different mechanisms at various steps in the apoptotic pathway to functionally block this cell death reaction (Hershberger et al., 1992 ; Clem & Miller, 1994 ; Clem et al., 1996 ; Manji et al., 1997 ; Miller, 1997 ; Seshagiri & Miller, 1997 ; Zoog et al., 1999 ).

Since the discovery of vAcAnh, a p35 mutant of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) inducing apoptosis in Sf21 cells (Clem et al., 1991 ), other reported cases of baculovirus-induced apoptosis included wild-type (wt) AcMNPV in SL2 (Chejanovsky & Gershburg, 1995 ) and CF-203 cells (Palli et al., 1996 ), p35 mutants of Bombyx mori nucleopolyhedrovirus in BmN cells (Kamita et al., 1993 ) and Heliothis armigera single capsid nucleopolyhedrovirus in Hi5 cells (Dai et al., 1999 ). However, the molecular mechanism(s) involved in apoptosis signalling is still unknown. Potential stimuli consist of shut-off of RNA synthesis, viral DNA replication and viral gene expression, such as the ie-1 gene (Prikhod’ko & Miller, 1996 ; Miller, 1997 ; Clem, 2001 ), and it is possible that several factors are involved in triggering cell death (LaCount & Friesen, 1997 ).

Our previous studies showed that wt AcMNPV induces apoptosis in the insect cell line Sl-zsu-1, derived from Spodoptera litura (Xie et al., 1988 ), at 8 to 12 h p.i. (Dai et al., 1998 ) and that the AcMNPV ie-1 gene was vital in cell death signalling (Zhang et al., 2002a ). Virus early replication events were demonstrated to be sufficient for leading apoptosis to its peak (Zhang et al., 2002a ). It has also been shown that S. litura larvae, from China, India and Japan, were nonsusceptible to AcMNPV infection per os (unpublished data). In order to test the hypothesis that apoptosis is a limiting factor for virus propagation in nonsusceptible host(s), we used haemocoel injections of AcMNPV budded virions (BV) in S. litura larvae.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell and virus.
S. frugiperda IPLB-Sf-9 (Sf9) cells were grown in TC-100 medium supplemented with 10% heat-inactivated foetal bovine serum. Monolayers were infected with wt AcMNPV E2 strain and the culture supernatant was collected 2 days p.i. Titres of AcMNPV were determined by standard plaque assay using Sf9 cells (O'Reilly et al., 1992 ).

{blacksquare} Insect and bioassay.
S. litura and S. exigua larvae were reared in the laboratory in individual cups on artificial diets (O'Reilly et al., 1992 ) at 27 °C under a 14/10 h light/dark cycle. Six dose rates of wt AcMNPV BV ranging from 2x101 to 2x106 p.f.u. per larva were diluted in complete TC-100 medium. Fourth instar larvae were inoculated by injection into the haemocoel (10 µl per larva, ten larvae were injected per dose rate). Mock-infected insects were injected with 10 µl TC-100 medium. Larvae mortalities were recorded every day. Larvae that failed to respond to agitation were considered dead.

{blacksquare} Tissue preparation and sectioning.
One S. litura larva was killed at 24, 48, 72, 96 and 120 h p.i. and fixed in Davidson’s AFA solution (Bell & Lightner, 1988 ) for 12 h. The larval abdominal segments were severed from other segments with a razor blade and fixed in Davidson’s AFA solution again for another 12 h. Following several rinses in distilled water, the larvae were dehydrated in graded ethanol to butanol–paraffin and embedded in paraffin. Sections were cut cross-sectionally at a thickness of 6 µm, stained using Hamm’s method (Hamm, 1966 ) and photographed.

{blacksquare} Apoptotic assays
(1) DNA fragmentation.
S. litura larval haemocytes were collected at 12, 24, 48, 72, 96 and 120 h p.i., washed with PBS (pH 7·2) buffer with 0·1% DTT and centrifuged for 1 min at 1000 g. The pellets were then incubated in lysis buffer (20 mM Tris–HCl, pH 8·0, 10 mM EDTA and 1% NP-40) for 5 min and the supernatants collected after being centrifuged at 1600 g for 5 min. SDS and RNase A was added to a final concentration of 1% and 5 µg/ml, respectively. After 2 h of incubation at 56 °C, the supernatants were digested with proteinase K at 37 °C for 2 h to a final concentration of 2·5 µg/ml. The DNAs were precipitated with ethanol, dissolved in TE and separated by electrophoresis on 1·5% agarose gels.

(2) In situ cell death detection.
This assay was performed using the In Situ Cell Death Detection kit, as described by the manufacturer (Boehringer Mannheim). Sections were deparaffinized, rehydrated and incubated with proteinase K (20 µg/ml in PBS, pH 7·2) for 30 min at room temperature. After being washed twice with PBS, the sections were incubated with 50 µl TUNEL (terminal dUTP nick end labelling) reaction mixture containing TdT and fluorescein–dUTP for 1 h at 37 °C and washed three times with PBS. Samples were treated with 50 µl Converter-AP for 30 min at 37 °C. Slides were rinsed three times in PBS at room temperature for 5 min, followed by the addition of NBT/BCIP solution and incubation at room temperature for 10 min. After washing with PBS, the sections were observed by light microscopy.

{blacksquare} Electron microscopy.
Haemocytes of S. litura larvae were fixed in 2·5% glutaraldehyde overnight at 4 °C, washed three times with PBS buffer and post-fixed in 1% osmium tetroxide in PBS buffer for 1 h at 4 °C. After fixing, haemocytes were washed three times with PBS buffer, dehydrated in graded ethanol and soaked in acetone. Infiltration was accomplished using Spurr’s resin in acetone (1:1) and haemocytes were embedded in fresh Spurr’s resin in gelatin capsules. Thin sections were stained with uranyl acetate and lead citrate and observed under a JEM-100CXII transmission electron microscope operating at 80 kV.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Pathology of S. litura larvae infected by wt AcMNPV
Direct inoculation of all doses of AcMNPV BV tested (2x101–2x106 p.f.u. per larva) into S. litura larval haemocoel were observed to result in death and the time from infection to death generally increased with increasing doses of BV (Table 1). In larvae injected with the highest dose of BV, growth was slowed down at 2 days p.i., feeding stopped at 3 days p.i. and death was observed at 5 days p.i. (Fig. 1). Mock-infected larvae began to pupate at 6 days after mock-infection.


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Table 1. Time to death of S. litura larvae injected with AcMNPV.

 


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Fig. 1. Morphological changes of S. litura larvae taken at days 0, 1, 2, 3, 4 and 5 post-injection with a high dose of AcMNPV BV (2x106 p.f.u. per larva). Mock-infected larvae are on the left.

 
Larvae injected with AcMNPV and those injected with the pathological virus SpltMNPV (Spodoptera litura multicapsid nucleopolyhedrovirus) (Pang et al., 2001 ) displayed different symptoms of infection. Cadavers did not melt, liquefy and melanize as usual when infected with wt SpltMNPV.

Haemocytes blebbing and nuclear fragmentation
Haemocyte samples taken every 12 h after virus inoculation showed similar blebbing of cell membranes at 24 h p.i. for the higher doses, ranging from 2x103 to 2x106 p.f.u. per larva of AcMNPV BV (Fig. 2A, panel 2, 2x106 p.f.u. per larva BV were injected). The number of apoptotic cells increased (Fig. 2A, panels 3–6) with time (48, 72, 96 and 120 h) and peaked at 120 h p.i. (ca. 60% cells underwent apoptosis). Haemocytes from larvae injected with TC-100 medium were normal (Fig. 2A, panel 1). At the lower doses of virus (2x101–2x103 p.f.u. per larva), the number of apoptotic cells peaked later (at 4–5 days p.i.). In both cases, some haemocytes showed the typical cytopathic symptoms of baculovirus infection of being swollen and round and less than 1% of these cells produced occlusion bodies.



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Fig. 2. Apoptosis in S. litura larvae haemocytes post-injection with AcMNPV. (A) Light micrographs (magnification x200) of cells harvested from larvae injected with 10 µl TC-100 medium (panel 1) or 10 µl AcMNPV (1x108 p.f.u./ml) (panels 2–6, at 24, 48, 76, 96 and 120 h p.i., respectively). (B) Fragmented DNA detection. Total cellular DNAs were extracted from haemocytes (300 µl lymph per lane, except 100 µl collected from dying larvae at the last day) at 12, 24, 48, 72, 96 and 120 h p.i. (lanes 2, 4, 6, 8, 10 and 12, respectively) and DNAs from mock-infected larvae were shown to the left of them as a control (lanes 1, 3, 5, 7, 9 and 11, respectively). DNA molecular mass markers (bp) are indicated at the left.

 
Oligonucleosome-sized ladders were observed after electrophoresis of total DNAs extracted from haemocytes injected with the highest dose of BV (2x106 p.f.u. per larva) (Fig. 2B). The DNA ladder pattern was detected first at 24 h p.i. and became more distinct after a longer time (Fig. 2B, lanes 4, 6, 8 and 10; lane 12, fragmented DNAs decreased due to less sample available). However, DNA fragmentation was not detected with DNAs extracted from the control (Fig. 2B, lanes 1, 3, 5, 7, 9 and 11) or infected insects at 12 h p.i. (Fig. 2B, lane 2).

Ultrastructure of haemocytes
Haemocytes from larvae inoculated with the highest dose of BV (2x106 p.f.u. per larva) and larvae from the mock-infection at 4 days p.i. were fixed and checked for apoptosis and virus replication under the transmission electron microscope. Definite ultrastructural changes in haemoctyes induced by apoptosis were observed. Characteristics of an early stage of apoptosis were evident, such as chromatin margination (Fig. 3B), chromatin condensation and nuclear disassembly (Fig. 3C). In Fig. 3(B, C), cell cytoplasm had become vacuolized. Structural changes of later stages of apoptosis, including blebbing of cell surface, formation of apoptotic bodies and accumulation of cell debris (probably due to disintegration of apoptotic cells) were observed (Fig. 3D). Mock-infected cells retained their normal structures (Fig. 3A).



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Fig. 3. Ultrastructure of haemocytes from S. litura larvae. Thin sections of cell pellets were stained with uranyl acetate and lead citrate and examined under a JEM-100CXII transmission electron microscope. (A) Mock-infected, magnification x10000. (B)–(F) Cells infected with AcMNPV, collected and fixed at 96 h p.i. Magnification: x10000 (B); x14000 (C); x4000 (D); x14000 (E); x27000 (F). Note that some vacuoles are visible in the cytoplasm in (B) and (C). Arrows point to virus particles. VS, virogenic stroma.

 
A small proportion of haemocytes (ca. 5%) showed typical events of productive virus infection. Dense virogenic stroma (VS) could be seen in the central region of the nucleus and high numbers of rod-shaped nucleocapsids were also seen in VS (Fig. 3E, F). However, no baculovirus occlusion bodies (OBs) were detected in these cells.

Apoptosis at the level of the organism
The TUNEL method in vivo was used to evaluate the extent of apoptosis induced in tissues other than in haemocytes. Nuclei in fat body cells were positively stained at early stages p.i. (Fig. 4B, 24 h p.i.). The nuclei in most other tissues, including fat body, epithelial and midgut cells, were positively stained in later stages of infection (Fig. 4C–F, 48, 72, 96 and 120 h). In late stages of virus infection (Fig. 4E, F), the cells did not keep an intact structure, cell membranes were blurred and the stained nuclei smeared together. Cell nuclei of mock-infected larvae were not stained at all (Fig. 4A).



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Fig. 4. In situ detection of DNA nick ends (fragmented DNA) by TUNEL exhibited positively staining nuclei in fat body cells, epithelial cells (B–F, larval sections prepared at days 1, 2, 3, 4 and 5 p.i.) and midgut cells (E) post-injection with AcMNPV BV, which are absent in mock-infected larvae (A).

 
Production of AcMNPV BV in the host S. litura larvae post-injection
In order to examine if production of in vivo progeny of AcMNPV BV was affected by larval cell apoptosis, larval haemolymph was collected every day post-injection at the highest dose of BV (2x106 p.f.u. per larva) and titrated in Sf9 cells. The level of BV yielded in S. litura larvae was very low on the first day p.i., about 5000-fold lower than the amount yielded in S. exigua larvae (Table 2). BV production in S. litura larvae kept increasing with time but it was still 200-fold lower than that in S. exigua larvae at 2 days p.i. (Table 2).


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Table 2. Comparison of the BV production levels in S. litura and S. exigua larvae p.i.

 
Histopathology of S. litura larva after AcMNPV infection
Larval tissues fixed daily p.i. for 5 days were checked for histopathological effects. Using Hamm’s stain, OBs could be detected in epithelial and fat body cells, with polyhedra stained red in the swollen nuclei (Fig. 5C, 48 h p.i.). Although more OBs were observed at 72, 96 and 120 h p.i. (Fig. 5D–F, respectively), less than 10% of cells were detected with OBs at 120 h p.i. (Fig. 5F). In Fig. 5(F), most cells underwent marked morphological changes, neighbouring cell membranes were no longer distinct and became blurred, probably due to cellular disintegration caused by apoptosis. The control larva tissues remained normal (Fig. 5A).



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Fig. 5. Histopathology of S. litura larvae. Sections were stained using Hamm’s method. Mock-infected larva (A) and larvae injected with the highest dose (2x106 p.f.u. per larva) of BV at 1, 2, 3, 4 and 5 days p.i. (B–F) are shown. Virus particles were stained red in the swollen nuclei (C–F).

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this study, we have demonstrated that in vivo apoptosis occurs in S. litura larvae upon inoculation with AcMNPV BV. The haemocytes of S. litura larvae injected with AcMNPV BV showed all the characteristics of apoptosis. These included both cell morphological changes, such as blebbing of cell surface, chromatin margination and condensation, vacuolization of the cytoplasm and formation of apoptotic bodies, and the molecular changes of intranucleosomal DNA fragmentation. The appearance of cell surface blebbing 24 h p.i. coincides with the beginning of DNA fragmentation. Apoptosis of other tissue cells has also been demonstrated as positive by TUNEL, a method to detect apoptotic cell death at a single-cell level (Gavrieli et al., 1992 ).

It is interesting that infection of S. litura larvae by wt AcMNPV produces much less progeny BV, compared with that in infected S. exigua larvae, a susceptible host for AcMNPV. In the former, only a small proportion of tissue cells, such as haemocytes, epithelial cells and fat body cells, produce OBs beginning from 48 h p.i., as shown by transmission electron and light microscopy. We supposed that the apoptosis occurring in S. litura larvae body cells may have disturbed virus replication, via self-damage of some cellular substructures, and resulted in this reduction in virus infectivity.

We have also noted that although wt AcMNPV BV infection is lethal to S. litura larvae when the virus is injected into the haemocoel, the cadavers do not melt, liquefy and melanize. These symptoms are similar to S. frugiperda larvae infected with vP35Z and vAcAnh. However, in the latter, a high dose of BV is needed to kill the S. frugiperda larvae and no consistent apoptosis has been observed (Clem & Miller, 1993 ). It is possible that certain tissue(s) involved in the melting process failed to yield necessary melting factors due to apoptosis. The cadavers remained intact, which could hinder the horizontal spread of baculovirus progeny.

AcMNPV-induced apoptosis showed a marked reduction in virus in vivo infectivity, which is most probably the reason that the spread of virus progeny is limited in its nonsusceptible host, S. litura. However, the mechanism by which AcMNPV, with a wide spectrum of host cells permissive for its productive replication (Carbonell et al., 1985 ; Vail & Jay, 1973 ), triggers S. litura apoptosis both in vivo and in vitro remains unknown. The AcMNPV p35 gene is well known for its anti-apoptotic response against host defences and is important for productive virus replication. However, P35 can only exert its function if present at the right time and in sufficient amounts. Previously, it had been reported that AcMNPV-induced SL2 cell apoptosis is inhibited by p35 overexpression but not when insufficiently expressed (Gershburg et al., 1997 ). In a previous study we also showed that the AcMNPV ie-1 gene induces apoptosis in Sl–zsu-1 cells when transfected alone (Zhang et al., 2002a ). Therefore, we suggested here that the ie-1 gene, together with other factors, may trigger apoptosis by interfering with the host cell cycle before p35 is fully expressed (Prikhod’ko & Miller, 1996 ; Gershburg et al., 1997 ).

When Sl-zsu-1 was coinfected with AcMNPV and wt SpltMNPV, SpltMNPV was capable of inhibiting AcMNPV-induced apoptosis and rescuing AcMNPV replication (Zhang et al., 2002b ). It follows that specific gene(s) in the SpltMNPV genome may have the potential to expand the host range of AcMNPV. Recently, the SpltMNPV genome has been sequenced and a p35 homologue (the p49 gene) and an iap gene were found (Pang et al., 2001 ). The P49 protein, sharing 30% identity with AcMNPV P35 and 79% identity with P49 of SpliMNPV (Spodoptera littoralis nucleopolyhedrovirus) (Du et al., 1999 ), may have a similar anti-apoptotic function to P35.

In conclusion, wt AcMNPV induced apoptosis and displayed reduced virus infectivity and virus propagation in S. litura larvae, indicating that apoptosis could represent a type of immune response of insects. Wt AcMNPV, together with its nonsusceptible host S. litura, constitutes an ideal model system to explore the specificity, coevolution and interrelationships between viruses and their hosts. Therefore, further studies on the possible role of apoptosis in limiting the host range of baculoviruses using this model system are warranted.


   Acknowledgments
 
We thank Dr D.J. Rae for her comments on the manuscript. This research was supported by the National Natural Science Foundation of China (nos. 39730030 and 39800092) and the National Major Basic Research Project ‘973’ of China (no. G2000016209).


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 6 June 2002; accepted 12 August 2002.