National Institute of Sericultural and Entomological Science, Tsukuba, 305-8634 Ibaraki, Japan1
Department of Chemistry for Materials2 and School of Medicine3, Mie University, Tsu, 514-8507 Mie, Japan
Author for correspondence: Wataru Mitsuhashi. Fax+81 298 38 6028. e-mail mitsuhas{at}nises.affrc.go.jp
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Abstract |
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Introduction |
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Similarly, polyhedrin-negative recombinant baculoviruses, which have been used in various scientific fields in recent years, are also rarely infectious to lepidopteran larvae through peroral inoculation, since, like NOVs, they are not occluded within polyhedra. Therefore, recombinant baculoviruses have to be injected into the insect haemocoel one by one to infect lepidopteran larvae with the viruses. This method is quite laborious when a large number of host insects is used. If polyhedrin-negative baculoviruses could become infectious via peroral inoculation by a novel method, this technique should be very useful for large-scale inoculation experiments.
Most entomopoxviruses (EPVs) form two distinctive types of proteinaceous structures, the spindle and spheroid. The spheroid contains virions. On the other hand, the spindle, composed mainly of the protein fusolin, has no virions (Dall et al., 1993 ). It has been revealed that EPV spindles possess the interesting function of acting as an enhancing agent for the infectivity of NPVs (polyhedra) in the same host insect (the armyworm, Pseudaletia separata) (Hayakawa et al., 1996
; Hukuhara et al., 1995
; Wijonarko & Hukuhara, 1998
). More recently, we indicated that spindles of Anomala cuprea entomopoxvirus (AcEPV) from the coleopteran A. cuprea greatly enhanced the infectivity of NPV (polyhedra) in the lepidopteran B. mori (Mitsuhashi et al., 1998
). These findings suggest that EPV spindles may enhance the infectivity of non-occluded forms of BmNPV, which are rarely infectious by peroral inoculation, in silkworm larvae. In addition, the infectivity of polyhedrin-negative recombinant baculoviruses, which do not produce polyhedra, may also be enhanced when administered with EPV spindles.
In the present study, we examined whether the peroral infectivity of wild-type NOVs and polyhedrin-negative recombinant viruses of BmNPV was enhanced when administered to silkworm larvae with AcEPV spindles. In addition, the capacity of AcEPV spheroids to enhance infectivity of NOVs and recombinant viruses was also assayed in silkworm larvae.
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Methods |
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BmNPV that has been maintained in our laboratory was also used. NOVs of BmNPV were collected from grassery juice (haemolymph of BmNPV-infected silkworm larvae) as follows. The grassery juice was centrifuged at 7100 g for 10 min to remove the haemocyte and occlusion bodies (polyhedra). The centrifugation was repeated twice. The supernatants containing NOVs were further filtered through a 0·45 µm membrane (Nihon Millipore).
A recombinant BmNPV producing prolixin-S (an anti-coagulant from the salivary gland of the blood-sucking bug Rhodnius prolixus) (Sun et al., 1996 ) used in this study was constructed as described by Maeda (1989)
with several modifications. The prolixin-S cDNA cloned in pBluescript II SK+ was excised by EcoRI digestion and ligated into the transfer vector pBM030 at an EcoRI site just downstream of the polyhedrin promoter in the positive orientation. This prolixin-SpBM030 construct (5 µg) and wild-type BmNPV T3 DNA (1 µg) were mixed with 8 µl Lipofectin reagent (Gibco-BRL) and co-transfected to 1x106 BmN4 cells as described by Kobayashi & Belloncik (1993)
. A polyhedrin-negative recombinant BmNPV was purified by plaque assay and prolixin-S production was confirmed by Western blotting with a rabbit antiserum raised against native prolixin-S (Sun et al., 1996
). The purified recombinant BmNPV was injected into and propagated in silkworm larvae. Free virions of the recombinant BmNPV that accumulated in haemolymph of the infected larvae were collected, centrifuged and filtered as described above. The filtration was performed to adjust infectivity to a similar level as that of the NOV suspensions used in this experiment.
Purification of spindles and spheroids.
Spindles and spheroids of AcEPV were collected and purified by a modification of the method of Mitsuhashi et al. (1997) . Briefly, dead larvae of A. cuprea infected with AcEPV were macerated and then filtered through cheesecloth to remove larval debris. The filtrate was subjected to differential centrifugation and the suspension was sonicated briefly.
Spindles were roughly separated from a sonicated mixture of spindles and spheroids by 6175% (w/v) sucrose density gradient centrifugation at 72200 g for 1 h at 5 °C in a Beckman SW28 rotor. The spindle suspension was purified by two further cycles of centrifugation of 6575% (w/v) sucrose density gradients and then two cycles of 7081% (w/v) sucrose density gradients and finally by potassium iodide density gradient centrifugation using a potassium iodide solution of density 1·36 g/ml according to the method of Bergoin et al. (1970) . The pellet of spindles was collected and washed with sterile distilled water several times to remove the potassium iodide. The spheroid suspension obtained from the first 6175% sucrose density gradient centrifugation was purified further by four 6575% (w/v) sucrose density gradient centrifugations and potassium iodide density gradient centrifugation using a potassium iodide solution of density 1·36 g/ml and spheroids were collected from the band of centrifugation and washed several times with sterile distilled water. Thus, the final spindle and spheroid suspensions were highly purified, demonstrating purities of 99·5% (content of about 0·5 % spheroids in comparison with the number of spindles) and 98·0% (content of about 2·0% spindles in comparison with the number of spheroids).
Enhancing experiments.
Silkworm larvae (B. mori, race Habataki) were reared on an artificial diet (Silkmate; Nihon-nosan Co. Ltd, Tokyo, Japan) at 26 °C and were used as 2nd to 5th instar larvae. A small piece of the artificial diet containing NOVs (or recombinant viruses) and spindles (or spheroids) at various concentrations was placed in a rearing box. Twenty larvae in the box were allowed access to the diet containing NOVs and spindles for 24 h (4th and 5th instar), 28 h (3rd instar) or 38 h (2nd instar), or the diet containing NOVs and spheroids for 24 h (3rd and 4th instar), at 26 °C from the first day of each instar, and thereafter they were reared on normal diet at 26 °C. In some cases, 10 or 15 larvae were used. Diets containing either NOVs (or recombinant viruses) or spindles (or spheroids) were also used as controls in every experiment. These larvae fed completely on a whole diet containing spindles (or spheroids) and/or NOVs (or recombinant viruses), so that numbers of spindles or spheroids per larva were estimated based on the mean amount of artificial diet consumed by larvae. Inoculated silkworm larvae were observed for a week to check for the appearance of characteristic symptoms of BmNPV disease.
The original NOV (or recombinant virus) suspension was mixed with an equal volume of the spindle (or spheroid) suspension. This first dilution was expressed as 10-1 not 100 to obtain the log(LD50) with greater reliability, so that the dilutions 10-2 to 10-5 represent 20- to 20000-fold dilutions of the original suspension. A constant amount (2nd instar, 0·1 ml; 3rd to 5th instar, 0·2 ml) of each dilution was included in each piece of artificial diet.
The infectivity index (-log LD50) of the original NOV suspension after filtration through 0·45 µm was estimated to be 5·10 when 5th instar larvae were inoculated by intracoelomic injection (5 µl per larva). In addition, the infectivity index (-log LD50) of the recombinant virus suspension after filtration (through 0·45 µm) was estimated to be 5·60 when 4th instar larvae were inoculated by intracoelomic injection (5 µl per larva).
Statistical analyses.
LD50 and enhancing index were analysed by the 95% confidence limit (CL) of the SpearmanKärber method (Finney, 1964 ).
Furthermore, as a statistical model for the mortality of silkworm larvae, the equation arcsine(p0·5)=SD+ND+SD*ND was used, where p represents the mortality of silkworm larvae based on the observed number of dead larvae, SD represents the main effects of the spindle (or spheroid) dose on the mortality of silkworm larvae and ND represents the main effects of the NOV (or recombinant virus) dose on the mortality of silkworm larvae. SD and ND are treated as continuous variables. Statistical tests were performed by using JMP (SAS Institute, 2000 ).
Detection of spheroids after feeding.
Spheroids in midgut contents and faeces were collected from silkworm larvae 24 h after inoculation of 2·0x106 spheroids per larva with the 10-1 NOV suspension in an artificial diet and were observed with an optical microscope (x400).
Whether spheroids (1·33x108/ml final concentration) dissolved in digestive fluid (1·5-fold dilution) collected from 5th instar larvae or not was recorded. As a positive control, polyhedra of BmNPV (about 1x108/ml final concentration) were used. After these mixtures were incubated for 1 h at room temperature, occlusion bodies were observed with an optical microscope (x400).
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Results |
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Statistical analysis revealed that the spindle dose had a significant effect on the infectivity of NOVs in any instar larvae examined. A significant interaction between the spindle dose and NOV dose was found in all instars except 3rd instar-2. The values of F and P for SD and SD*ND in respective instars are as follows: 2nd instar, for SD, F=59·86 and P<0·0001 and for SD*ND, F=18·13 and P=0·0028; 3rd instar-1, F=138·79 and P<0·0001, F=23·54 and P=0·0019; 3rd instar-2, F=48·74 and P=0·0004; F=1·07 and P=0·3405; 4th instar, F=69·22 and P<0·0001, F=10·24 and P=0·0151.
Enhancement of infection with recombinant BmNPV by spindles
The enhancement of peroral infection with recombinant viruses by AcEPV spindles was assayed by using 3rd and 4th instar silkworm larvae. The results are shown in Table 2. Recombinant viruses alone were not infectious to either instar of silkworm larvae. However, the recombinant viruses became very infectious when they were administered with spindles. The logarithmic enhancing index of recombinant virus infectivity with spindles was as high as 3·33, which was almost the same level as that observed for NOV infectivity (Table 2
; third instar). No polyhedra were observed in any of the randomly selected dead larvae infected with recombinant viruses.
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Enhancement by spheroids
The potential enhancement of peroral infection with NOVs and recombinant viruses by AcEPV spheroids was assayed by using 3rd and 4th instar silkworm larvae. The results are shown in Table 3. No enhancement of peroral infection with either type of virus was observed in any assay. Although several larvae died of BmNPV disease when inoculated with a large number of viruses (10-1 dilution), that may have been caused by accidental infection with these viruses through the injured skin of leg tips and not per os in these assays. However, these results do not affect the calculated enhancing index of the spheroids.
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Fate of spheroids after feeding
Many AcEPV spheroids fed to silkworm larvae were observed in the midgut contents and faeces and were apparently unchanged in appearance.
Whether spheroids dissolved in the digestive fluid or not was also recorded. Spheroids did not appear to dissolve in the digestive fluid, while polyhedra of BmNPV dissolved completely over 1 h.
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Discussion |
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Hukuhara et al. (1995) observed by immunoelectron microscope analysis that the factor in Pseudaletia separata entomopoxvirus (PsEPV) that enhances the infectivity of an NPV in the armyworm, Pseudaletia separata, is present in the virions within the spheroid as well as the spindles, and PsEPV spheroids have been reported to harbour a strong enhancing ability akin to that of PsEPV spindles (Wijonarko & Hukuhara, 1998
). The fact that spheroids did not have an enhancing effect on the results of the present study may be attributed to the absence or very small number of virions liberated from spheroids. The reason for this is that AcEPV spheroids appeared not to dissolve in the digestive fluid of silkworm larvae; spheroids intact in appearance were usually detected in the midguts and faeces. Therefore, virions with a possible enhancing effect may not have been liberated from spheroids.
The mechanism by which AcEPV spindles enhance peroral infectivity of BmNPVs (NOVs and OVs) is still unknown. However, the present study suggests that spindles increase virus efficacy at some point after (not before) the dissolution of polyhedra (OVs), since a high level of enhancing ability (3 to 4 as log) was observed in both NOVs (this study) and OVs (Mitsuhashi et al., 1998 ). Therefore, the increased efficacy may be attributed to some change in infection sites in the midgut. For instance, spindles may disrupt the peritrophic membrane of the midgut to allow the virions to penetrate in a more efficient manner. Wang & Granados (1997)
revealed that an intestinal mucin is the target substrate for a baculovirus (granulovirus) enhancin. Another possibility is that spindles may contact insect midgut cells and alter these cells to be more susceptible to infection. Hukuhara et al. (1998)
also suggested that enhancing factors may relate to the process of fusion between NPV virions and insect cells, from an experiment using armyworm cells in culture.
Enhancing agents other than EPV spindles have also been documented. Dougherty et al. (1995) reported that the fluorescent brightener calcofluor (white M2R) increased the efficacy of NOVs and virions that were released from polyhedra by an alkali treatment, in addition to polyhedra of NPVs of the gypsy moth Lymantria dispar.
The finding that peroral infection with NOVs is enhanced by AcEPV spindles suggests that peroral infection of silkworm larvae with polyhedrin-negative recombinant baculoviruses might also be enhanced by spindles. As we assumed, peroral infection with a polyhedrin-negative recombinant BmNPV was enhanced at a level similar to that observed with NOVs. This is the first report of the enhancement of peroral infection with recombinant baculovirus by EPV spindles. Our novel method of enhancing peroral infectivity of polyhedrin-negative recombinant baculovirus by using AcEPV spindles should be very useful for large-scale inoculation experiments or for various kinds of biofactories (mass production) using recombinant viruses, since it is easy to inoculate these recombinant viruses to a large number of host insects.
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Acknowledgments |
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References |
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Received 17 July 2000;
accepted 20 October 2000.
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