Peroral infectivity of non-occluded viruses of Bombyx mori nucleopolyhedrovirus and polyhedrin-negative recombinant baculoviruses to silkworm larvae is drastically enhanced when administered with Anomala cuprea entomopoxvirus spindles

Y. Furuta1, W. Mitsuhashi1, J. Kobayashi2, S. Hayasaka1, S. Imanishi1, Y. Chinzei3 and M. Sato1

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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Non-occluded viruses (NOVs) of Bombyx mori nucleopolyhedrovirus (BmNPV) are poorly infectious to silkworm larvae when administered by peroral inoculation, although they are highly infectious when injected into the insect haemocoel. In the present study, it is demonstrated that NOVs of BmNPV became highly infectious even through peroral inoculation when administered with spindles (proteinaceous structures) of Anomala cuprea entomopoxvirus (AcEPV). Marked enhancement of peroral infectivity of NOVs by AcEPV spindles (nearly 1000-fold higher in the strongest case) was observed in all growth stages of silkworm larvae tested (2nd to 5th instar). Similarly, peroral infectivity of polyhedrin-negative recombinants of BmNPV, which do not produce polyhedra, was also enhanced remarkably by AcEPV spindles. In contrast, spheroids (proteinaceous structures containing AcEPV virions) did not enhance the peroral infectivity of either NOVs or the recombinant BmNPV in silkworm larvae.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Nucleopolyhedroviruses (NPVs), including Bombyx mori NPV (BmNPV), comprise a group of baculoviruses that are the most common and most widely studied group of viruses pathogenic to insects. NPVs have two phenotypes as infectious agents, a non-occluded virus (NOV) and an occluded virus (OV) (polyhedron-derived virus) (Tanada & Kaya, 1993 ). The OVs are the main infectious elements for horizontal transmission through the midgut of a susceptible host. In contrast, NOVs are rarely infectious through the midgut by peroral inoculation, although they are highly infectious when inoculated into the insect haemocoel (e.g. Kawarabata, 1974 ). Furuta (1972) reported that the NOVs of BmNPV did not infect silkworm larvae by peroral inoculation even though they were infectious when introduced via an intracoelomic injection. Similarly, Volkman & Summers (1977) showed that the infectivity of NOVs of Autographa californica NPV by peroral ingestion was approximately 104-fold lower than when NOVs were inoculated by intracoelomic injection.

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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses.
The AcEPV used in this experiment was isolated from a site in Shizuoka-ken, Japan, and was propagated in A. cuprea (Coleoptera: Scarabaeidae) larvae reared in our laboratory by feeding AcEPV spheroids.

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-S–pBM030 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.

{blacksquare} 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 61–75% (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 65–75% (w/v) sucrose density gradients and then two cycles of 70–81% (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 61–75% sucrose density gradient centrifugation was purified further by four 65–75% (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).

{blacksquare} 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).

{blacksquare} Statistical analyses.
LD50 and enhancing index were analysed by the 95% confidence limit (CL) of the Spearman–Kärber 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 ).

{blacksquare} 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).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Enhancement of infectivity of BmNPV NOVs by spindles
The enhancement of peroral infection with NOVs by EPV spindles was assayed by using 2nd to 5th instar silkworm larvae. The results are shown in Table 1. NOVs alone were not infectious to any instar of silkworm larvae. In contrast, NOVs became highly infectious when they were administered with spindles, so that even a highly diluted NOV suspension (10-3) infected all instars of silkworm larvae. In the experiment with the 2nd instar larvae, consumption of 1x106 or 1x105 spindles per larva resulted in a remarkable enhancement of the infectivity of NOVs, with logarithmic enhancing indices of more than 3·0 (infectivity more than 1000-fold higher than in larvae without spindles) or 2·25, respectively. Similar enhancements were observed in the experiments with 3rd and 4th instar larvae fed with more than 2x105 (3rd instar) or 4x105 spindles (4th instar). An experiment that used NOV filtration samples other than those of the above-mentioned three trials revealed a similar level of enhancement (Table 1; 3rd instar-2). In experiments with 5th instar larvae, only 10 larvae were used in each bioassay, but similar results were obtained (data not shown).


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Table 1. Enhancement of the peroral infectivity of BmNPV NOVs to silkworm larvae by feeding spindles of AcEPV

 
In every experiment, NPV symptoms appeared in larvae 3–5 days after exposure to the infectious materials. No disease occurred in the larvae inoculated with spindles alone and these larvae grew normally, as described in a previous report (Mitsuhashi et al., 1998 ); these results show that AcEPV spindles are not toxic to larvae.

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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Table 2. Enhancement of the peroral infectivity of a polyhedrin-negative recombinant BmNPV to silkworm larvae by feeding spindles of AcEPV

 
Statistical analysis revealed that the spindle dose had a significant effect on the infectivity of recombinant viruses in both instars examined, while no significant interactions between the spindle dose and recombinant virus dose were found. The values of F and P of SD and SD*ND in respective instars are as follows: 3rd instar, F=33·67 and P=0·0007, F=0·10 and P=0·7621; 4th instar, F=57·45 and P=0·0006, F=4·26 and P=0·0939.

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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Table 3. Enhancement of the peroral infectivity of BmNPV NOVs (wild-type) or polyhedrin-negative recombinant BmNPV to silkworm larvae by feeding spheroids of AcEPV

 
Statistical analysis revealed that the spheroid dose did not have a significant effect on the infectivity of NOVs or recombinant viruses in either instar examined. Also, no significant interactions between the spheroid dose and NOV or recombinant virus dose were found. The values of F and P of SD and SD*ND in respective instars are as follows: 3rd instar (NOV): F=0·11 and P=0·7529, F=0·18 and P=0·6872; 3rd instar (recombinant virus), F=0·00 and P=0·9842, F=1·45 and P=0·2737; 4th instar (NOV), F=0·16 and P=0·7030, F=0·31 and P=0·6005; 4th instar (recombinant virus), F=1·03 and P=0·3491, F=0·71 and P=0·4314.

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.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In the present study, NOVs of BmNPV were found to be non-infectious to silkworm larvae by peroral inoculation, as described previously (Furuta, 1972 ). In contrast, NOVs became highly infectious to larvae by peroral inoculation when administered together with AcEPV spindles, indicating that AcEPV spindles greatly enhance the peroral infectivity of NOVs. Although the purified spindle suspension used in this experiment still contained a small number of spheroids (about 0·5% of the number of spindles), results of experiments with purified spheroid suspensions ruled out completely the possibility that enhancement occurred because of the spheroids in the spindle suspension. As shown in Table 3, spheroid suspensions did not enhance the peroral infectivity of NOVs in any bioassay with 3rd and 4th instar larvae. Thus, we verified that AcEPV spindles greatly enhance peroral infectivity of BmNPV NOVs in silkworm larvae, but that AcEPV spheroids do not. This is the first report of the enhancement of peroral infection with NPV NOVs by EPV spindles.

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.


   Acknowledgments
 
We sincerely thank Dr Kohji Yamamura (NIAES, Japan) for his helpful suggestions regarding statistical analyses.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Bergoin, M., Veyrunes, J.-C. & Scalla, R.(1970). Isolation and amino acid composition of the inclusions of Melolontha melolontha poxvirus. Virology 40, 760-763.

Dall, D., Sriskantha, A., Vera, A., Lai-Fook, J. & Symonds, T.(1993). A gene encoding a highly expressed spindle body protein of Heliothis armigera entomopoxvirus. Journal of General Virology 74, 1811-1818.[Abstract]

Dougherty, E. M., Guthrie, K. & Shapiro, M.(1995). In vitro effects of fluorescent brightener on the efficacy of occlusion body dissolution and polyhedral-derived virions. Biological Control 5, 383-388.

Finney, D. J. (1964). Statistical Methods in Biological Assays, 2nd edn. London: Charles Griffin.

Furuta, Y. (1972). The infectivity-value of polyhedra and nonoccluded virus in hemolymph collected from silkworm larvae infected with BmNPVs. Acta Sericologica 82, 33–40 (in Japanese).

Hayakawa, T., Xu, J. & Hukuhara, T.(1996). Cloning and sequencing of the gene for an enhancing factor from Pseudaletia separata entomopoxvirus. Gene 177, 269-270.[Medline]

Hukuhara, T., Yano, K., Xu, J., Tomita, M. & Miyajima, S.(1995). Detection of a virus enhancing factor in inclusion bodies of an entomopoxvirus by immunoelectron microscopy. Journal of Invertebrate Pathology 65, 315-317.

Hukuhara, T., Hayakawa, T. & Wijonarko, A. (1998). The mode of action and transgenesis of a virus enhancing factor of an entomopoxvirus. In Proceedings of the VIIth International Colloquium on Invertebrate Pathology and Microbial Control, pp 7–8. Sapporo, Japan.

Kawarabata, T.(1974). Highly infectious free virions in the hemolymph of the silkworm (Bombyx mori) infected with a nuclear polyhedrosis virus. Journal of Invertebrate Pathology 24, 196-200.[Medline]

Kobayashi, J. & Belloncik, S.(1993). Efficient lipofection method for transfection of the silkworm cell line, NISES-BoMo-15AIIc, with the DNA genome of the Bombyx mori nuclear polyhedrosis virus. Journal of Sericultural Science of Japan 62, 523-526.

Maeda, S.(1989). Gene transfer vectors of a baculovirus, Bombyx mori nuclear polyhedrosis virus, and their use for expression of foreign genes in insect cells. In Invertebrate Cell System Applications , pp. 167-181. Edited by J. Mitsuhashi. Boca Raton, FL:CRC Press.

Mitsuhashi, W., Saito, H. & Sato, M.(1997). Complete nucleotide sequence of the fusolin gene of an entomopoxvirus in the cupreous chafer, Anomala cuprea Hope (Coleoptera: Scarabaeidae). Insect Biochemistry and Molecular Biology 27, 869-876.[Medline]

Mitsuhashi, W., Furuta, Y. & Sato, M.(1998). The spindles of an entomopoxvirus of Coleoptera (Anomala cuprea) strongly enhance the infectivity of a nucleopolyhedrovirus in Lepidoptera (Bombyx mori). Journal of Invertebrate Pathology 71, 186-188.[Medline]

SAS Institute (2000). JMP User’s Guide, version 4. Cary, NC: SAS Institute.

Sun, J., Yamaguchi, M., Yuda, M., Miura, K., Takeya, H., Hirai, M., Matsuoka, H., Ando, K., Watanabe, T., Suzuki, K. & Chinzei, Y.(1996). Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus. Thrombosis and Haemostasis 75, 573-577.[Medline]

Tanada, Y. & Kaya, H. (1993). Insect Pathology. New York: Academic Press.

Volkman, L. E. & Summers, M. D.(1977). Autographa californica nuclear polyhedrosis virus: comparative infectivity of the occluded, alkali-liberated, and nonoccluded forms. Journal of Invertebrate Pathology 30, 102-103.[Medline]

Wang, P. & Granados, R. R.(1997). An intestinal mucin is the target substrate for a baculovirus enhancin. Proceedings of the National Academy of Sciences, USA 94, 6977-6982.[Abstract/Free Full Text]

Wijonarko, A. & Hukuhara, T.(1998). Detection of a virus enhancing factor in the spheroid, spindle, and virion of an entomopoxvirus. Journal of Invertebrate Pathology 72, 82-86.[Medline]

Received 17 July 2000; accepted 20 October 2000.



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