Archives of insect Biochemistry and Physiology 22:345-356 (1993) Insecticidal Potential of the Insect Parvovirus GmDNV Jacov Tal and Tipvadee Attathom Department of Virology, Faculty of Health Sciences, Ben-Gurion University of the Negeu, Beer-Sheva, Israel (J.T.); Department of Entomology, Kdsetsart University, Kampaengsaen Campus, Nakhonpathom, Thailand (T.A.) The insecticidal potential of Galleria mellonella densovirus (CmDNV) in third, fourth, and fifth instar larvae of the host, the greater wax moth, as a step toward the construction of a molecular vector for the introduction and expression of foreign genes in the larvae of these insects was studied. Third instar larvae are most susceptible to GmDNV. Viral RNA synthesis is more rapid in this stage and slowest in the fifth instar. Infection of prepupae by intradermic injection or by horizontal spread inhibited pupation. G m D N V D N A i s also infectious when introduced as a calcium phosphate precipitate. The t w o putative viral promoters were shown to be capable of driving the expression of the reporter gene chloramphenicol acetyltransferase (CAT) i n DNA-injected larvae. o 1993 Wiley-Liss, Inc. Key words: densovirus, larvacidal activity, gene expression INTRODUCTION The family Parvoviridae contains three genera: autonomously replicating parvoviruses, helper dependent parvoviruses (AAV*)and insect parvoviruses (DNV) [l].Members of the first two genera have a widespread distribution among warm-blooded animals, whereas the densoviruses have been isolated from various insect species. The two mammalian genera differ from each other Acknowledgments: We thank Dr. Peter Tijssen for the CrnDNV clone and Drs. Michael Friedlander and Alan H. Mehler for reviewing this manuscript. This work was supported by grants from the U.S. Agency for International Development (AID-CDR) (to T.A. and J.T.), and from the National Council for Research and Development, Israel, and the GSF, Miinchen, Germany (to J.T.). Received February 18, 1992; accepted April 9, 1992. Address reprint requests to Dr. Jacov Tal at Department of Virology, Faculty of Health Sciences, Ben-Curion University of the Negev, Beer-Sheva 84105, Israel. *Abbreviations used: AAV = adeno-associated virus; Ac-CoA = acetyl coenzyme A; CAT = chlorarnphenicol acetyltransferase; DEPC = diethyl pyrocarbonate; DNV = densonucleosisvirus; GIT = guanidine isothiocyanate; ITR = invertedterminal repeat; kb = kilobases; NS = nonstructural; O R F = open reading frame; RF = replicative form; SDS = sodium dodecyl sulfate; VP = viral protein. 0 1993 Wiley-Liss, Inc. 346 Tal and Attathom in the requirement of AAV for a helper virus, adenovirus or herpes simplex virus [1,2]. Current knowledge of densoviruses, although still limited, reveals that they combine some features that are characteristic of either AAV or the autonomous parvoviruses. The first densovirus, GmDNV, was isolated from larvae of the greater wax moth, Galleria mellonella . Since then, densoviruses have been isolated from Lepidoptera, Diptera, Orthoptera, Dictyoptera (Blattaria) and Odonata and to date over 20 densovirus species are known . Some of them have a wide host range but others exihibit species specificity so narrow as to infect one host only. They also differ in their pathogenicity and tissue specificity, in that some can cause an extensive infection in most or all tissues, whereas the replication of others is restricted to one tissue within the insect . The present studies involve GmDNV, which can replicate in all larval tissues except for the midgut [5,6]. Like all parvoviruses, the DNV virion is icosahedral, 20-24 nm in diameter. It contains DNA and protein but no lipids, and thereby is resistant to high temperatures and organic solvents. The genome is a 5.8 kb long, singlestranded DNA molecule, with identical 0.44 kb long ITR on both ends . Upon entering the insect cell, the viral DNA is first converted to a doublestranded RF DNA. Viral replication, including the assembly of virions, takes place entirely in the cell nucleus [8,9]. Each DNA strand is packaged into separate virion particles [lo]. The four structural proteins of GmDNV possess common sequences [ll].Their stoichiometry in larvae of early and late instars, however, is completely different, indicating that genome expression of DNV is differently regulated during insect development [111. In Figure 1, the putative genomic organization of GmDNV is compared with that of a representative vertebrate parvovirus, MVM. The GmDNV organization is deduced from the nucleotide sequence of the DNA  and from physical mapping (0. Gross and J. Tal, unreported data). In vertebrate parvoviruses all the viral RNAs (solid lines) and proteins (hatched boxes) are encoded on one strand of the RF DNA. The left half of the genome encodes MVM NS promoter loon 2000 3000 4000 5ooo YPptumorer GmDNV NS YP PVP Fig. 1. Comparative organization of M V M (a mammalian parvovirus) and CmDNV genomes. The organization of GmDNV DNA is putative. RNA transcripts are drawn in solid lines, viral proteins in hatched boxes; arrows designate polyadenylation sites. See text for more details. GmDNV as Insecticide 347 the nonstructural regulatory proteins and the right half encodes the viral structural proteins. To expand their limited coding capacity, mammalian parvoviruses exploit a variety of strategies such as multiple promoters, alternative splicing and proteolyticcleavage . In addition, the structural and nonstructural transcription units are partially overlapping. Two densoviruses, those of Bombyx mori [12,13] and Aedes aegypti  also have similar genomic organization. In contrast, the nucleotide sequences of GmDNV and of its related Junonia coeniu densovirus (not shown; M. Bergoin, personal communication) reveal a completely different genomic organization, in which both strands of the replicative form DNA contain open reading frames. Northern blot hybridization of RNA from GmDNV-injected larvae revealed five polyadenylated, virus-specific transcripts with sizes ranging from 1.8 kb to genome size. Physical mapping of GmDNV RNA indeed showed that the two major transcripts (1.8 and 2.4 kilobases) map to the "left" and "right" halves of the viral DNA, respectively, and hybridization with unidirectional singlestranded probes showed that these transcripts have opposite orientations. In vitro translation of hybrid-selected 2.4 kb message yielded four polypeptides which comigrated with the four viral structural proteins. In vitro translation of the 1.8 kb message yielded three polypeptides which were not found in virion extracts and thus seem to be viral nonstructural proteins (0.Gross and J. Tal, unreported data). Initially, this transcriptional organization seems to be a major deviation from that found in all mammalian parvoviruses. However, as Figure 1 demonstrates, the basic organization of all parvoviruses in which the "left" and "right" ORFs encode the nonstructural and structural proteins, respectively, is being conserved, albeit on different DNA strands. Densoviruses are highly infectious to their insect hosts but their use as insecticides has so far been very limited. In Columbia, extracts of DNV-infected larvae were used to control the disease of palm trees caused by Sibine fUscu . In the Ivory Coast, DNV of Casphalia extranea was used to control disease in oil palm and coconut trees . In this paper we investigated various parameters which affect the insecticidal potential of GmDNV, and describe our basic approach towards the construction of a GmDNV based vector. MATERIALS AND METHODS Larvae and virus. Galleria mellonella larvae were grown in a Gerber baby food (mixed cerea1):honey:glycerol:watermixture at the ratios of 200 g: 50 g:50 ml:50 ml, respectively, in a humidified incubator at 30°C. Larvae were either injected with virus, using a syringe with 2 PI pulse delivery mechanism (Hamilton Company, Reno, NV), or infected by incubating them in the presence of diseased larvae. To prepare GmDNV stock virus, larvae were infected with virus and then left to grow for 8 to 10 days in growth food. Dead larvae were stored frozen until use. To extract virus, larvae were suspended in TE buffer (20 nM Tris, pH 8, 2 mM EDTA) at a ratio of 200 to 400 mg/ml, placed on ice and disrupted in a tissue homogenizer (Polytron, Luzern, Switzerland, Model PT K with a PTAlOS aggregate) at full speed for 2 min. The virus was purified as published [7,16]. 348 Tal and Attathom Viral DNA clone. A 5.4 kbp BamHI-BamHI fragment of viral DNA, containing the entire coding region of the virus, was cloned into pUC18 vector. The BamHI sites reside within the inverted terminal repeats, 173 nt from the genome termini (P. Tijssen, personal communication). Preparation of RNA from infected larvae. Infected larvae, 48 h postinfection, were first submerged in a fresh suspension of DEPC 0.5% in water for 30 to 60 min, then frozen in liquid nitrogen. The DEPC was highly effective in reducing ribonuclease activity during RNA extraction. To prepare RNA, the larvae were dropped into a solution of GIT buffer (4 M GIT, 25 mM sodium acetate, pH 6, and 0.1 M p-mercaptoethanol), at a ratio of 100-200 mg larval weight per ml, and homogenized in a Polytron homogenizer under the same conditions as those described for virus preparation, The homogenate was clarified by filtration once through glass wool, layered on top of a 1.7 ml, 5.7 M CsCl cushion and centrifuged for 12 h in a SW 50.1 rotor (35,000 RPM at 20°C). The RNA pellet at the bottom of the tube was washed once with GIT buffer, then resuspended in H a and the concentration was determined. Initial preparations included a phenol extraction step after the GIT buffer wash, which was later omitted from the procedure. Poly (A)-containingRNA was isolated by chromatography through an oligo-dT-cellulosecolumn. Transient expression assays. A calcium precipitate of GmDNV-CAT DNA was prepared as described by Dubenski et al. . The precipitate was dissolved in sterile double distilled water and injected into larvae at 1 to 10 pg per 200 mg larval weight. Six days later larvae were suspended in 0.25 M Tris-HC1pH 8 (200 mg/ml, total volume), minced in Polytron homogenizer as described and centrifuged in a microcentrifugefor 10 min. The upper aqueous layer was assayed for CAT activity according to Gorman et al. . Size analysis of viral RNA. Formaldehyde denaturing gel electrophoresis of RNA and blotting to nitrocellulosewere done as described [21,23]. The filter was baked in a vacuum oven (2 h, SOOC) and h bridized with cloned GmDNV DNA or fragment, as specified, labeled with % by random prime-mediated incorporation  to 1-3 x lo9 cpmlpg, lo7 cpm per 100 cm', washed and autoradiographed with Agfa Curix RPM2 X-ray film. Y RESULTS Transcription of the GmDNV Genome in Galleria mellonella Larvae A major impediment to the study of densovirus gene expression is the absence of a tissue culture host for the virus. Therefore, the experiments described below were done with larvae injected with CsC1-purified GmDNV (10 ng virus/200 mg larva). One puzzle that emerges from the GmDNV sequence is the structures of the nonstructural and structural promoters. Usually we expect different regulation elements to operate in the expression of structural and nonstructural (regulatory) promoters, but computer analysis of the GmDNV sequence shows that the two putative promoters are embedded in the inverted terminal repeat, and this means that the upstream sequences of both are identical. To find out if the two promoters are regulated differently in GmDNV, total RNA was extracted from injected larvae and GmDNV as insecticide - 0 1 2 3 4 5 DAYS POSTINFECTION 349 6 Fig. 2. RNA synthesis in CmDNV-injected larvae. Third, 4th and 5th instar larvae (marked 111, IV and V, respectively), injected with virus (10 p,g virus per 200 mg larvae)were frozen at the indicated days. RNA from each time point was prepared, electrophoresed in formaldehyde gels, blotted onto nylon membranes and hybridized to 32P-labeledGmDNV DNA as described [211. After exposure to X-ray films, the 1.8 and 2.4 kb bands were individually excised and counted in a scintillation counter. Each time point represents 6 larvae weighing approximately 1.5 g. Filled symbols, 1.8 kb RNA; open symbols, 2.4 kb RNA. Larval stages were determined by the width of the head capsule. analyzed by Northern blot hybridization. The 1.8 and 2.4 kb bands were cut and counted in a scintillation counter. As shown in Figure 2, the relative abundance of the nonstructural mRNA remains steady throughout the infection, while the structural message increases at least twofold between day 1 and day 4. Insecticidal Potential of GmDNV Viral insecticides are slow to act and their killing efficiency depends highly on a variety of factors such as environmental conditions, method of application, the developmental state and behavioral traits of the insect, etc. Figure 3 is a demonstration that the 3rd instar of G. mettonella is the most vulnerable to killing by GmDNV. When virus is injected into larvae at a constant weight per weight ratio and the injected larvae are incubated under identical conditions, the killing of 3rd instar larvae is at least twice as fast as the killing of 4th and 5th instar larvae. These data suggest that virus replication is more efficient in 3rd instar larvae than in later stages. To examine this, replication of viral DNA and RNA in different stages of G. mellonella was studied. As expected, the most efficient synthesis of viral RNA (Fig. 4) and viral DNA (not shown) was found in 3rd instar, lower in 4th) and at least twofold lower in 5th instar. We next examined the effect of virus infection on the pupation of G. mellonella. Three groups of 20 prepupae each were used. The larvae in one group were incubated in the presence of diseased larvae; larvae from the second group were injected with GmDNV as described, and those in the third group were left untreated. All 3 groups were incubated in the presence of wax sheets as the sole source of food and pupated and dead larvae were counted daily. The results of this experiment (Fig. 5) show that pupation is inhibited 350 Tal and Attathom Fig. 3. Larvacidal activity of GmDNV. CrnDNV-injected larvae were incubated in the presence of wax sheets and mortality was recorded daily. Open circles, 5th instar; filled squares, 4th instar; filled circles, 3rd instar larvae. The results are an average from three separate experiments, each involving 60 larvae (20 from each instar). 400 I 3rd instar 4th instar ISthtnstar 300 z - F2 Hock i n f e c t e d 0 - +G Q E N ar 200 E= m 2 > I 1 00 n 1 3 5 7 DAYS POSTINFECTION Fig. 4. CmDNV RNA synthesis during infection. CmDNV larvae (20 from each instar; mock infected larvae were from 3rd instar) were frozen to -70°C at the indicated times after injection. RNA was extracted, treated with RNase-free DNase (Boehringer Mannheim, Germany) and quantitated spectrophotometrically. Samples from each extract containing identical RNA quantities were blotted onto nylon filters and hybridized with 32P-labeled CmDNV RNA. The blots were counted in a scintillation counter. GmDNV as Insecticide 351 0 Drrdlrrvre b p r t e d larvae: w Unlntcctcd Injected Horizontal infection L 6 3 5 7 11 DAYS POSTINFECTION Fig. 5. Inhibition of pupation by GmDNV. Prepupae larvae were either injected with CmDNV, incubated in the presence of diseased larvae or left untreated, and then incubated in bottles containing food. Mortality and pupation were monitored daily. by GmDNV, administered by injection or spread horizontally. Furthermore, the mortality rate in both groups was loo%, as pupating larvae did not produce moths and larvae which did not pupate (or pupated partially) eventually died. Development of Densovirus Vectors As stated, the killing rate of all insect viruses is slow, compared with chemicals. One approach to overcome this problem is the development of genetically engineered insect viruses with enhanced killing potential. This can be accomplished by substituting sections of the virus genome by genes with high insecticidal potential. Figure 6 shows our view of two prototype vectors, designed for transient expression of foreign genes in insects. In the structure at the top, a foreign gene is inserted in place of the structural genes, under the control of the authentic GmDNV VP promoter, which is embedded in the right inverted terminal repeat. The viral NS genes, shown to be responsible for a number of regulatory processes in the virus life cycle, is preserved. Based on the analogy to mammalian parvoviruses, the NS region in this structure is expected to promote DNA replication and to regulate expression from the VP promoter. In the bottom structure, the VP promoter is replaced either by a promoter that is responsive to environmental conditions, such as heat, or by a cellular promoter that is responsive to developmental processes in the insect, In this construct, the role of the NS region is to promote DNA replication. 352 Tal and Attathorn ** nh NS promoter I I Foreign promoter Fig. 6. Structures of prototype CmDNV vectors. The long horizontal arrows denote RNA transcripts. See text for details. Fig. 7. Expression of CAT in transjected larvae. Larvae were transjected with 2 pg of CAT vectors and allowed to grow in normal food. Four days later, the larvae were homogenized in a Dounce homogenizer in 0.25 M Tris-HCI buffer, pH 7.8, and the homogenate from which debris was removed by extensive centrifugation was assayed for CAT activity. The numbers at the bottom indicate the numbers of micrograms of vector DNA injected, and the numbers at the top indicate the percentage of conversion of CoA to its acetylated forms as determined by cutting and counting the spots in a scintillation counter. C, mock transjected larva. GmDNV as Insecticide 353 To examine the expression of the viral promoters in larvae, we have constructed two ”prototype vectors” in which the bacterial gene CAT was inserted under the control of the left and right GmDNV promoters (NS-cat and VP-cat, respectively), The CAT-containing plasmids were introduced into larvae using the “transjection” method described by Dubensky et al. . Six days later the larvae were homogenized in 0.25 M Tris-HC1, pH 7.8, and CAT expression in the debris-free homogenate was assayed. CAT expression from both promoters is well above the endogenous acetylatingactivity in the larvae, indicating that they are active in larvae (Fig. 7). DISCUSSION Viruses are naturally occurring vectors, which can be engineered to introduce foreign genes into cells. Eukaryotic viral vectors are being used to introduce foreign genes into the host cells, with the purpose of expressing them or introducing them into the host chromosome. The most commonly used insect viral vector, the baculovirus vector system, has been used to express a wide variety of foreign genes in insect cell lines. Despite its large genome (over 100 kb), baculovirus is the vector of choice for expression purposes because of the powerful polyhedrin promoter . In this paper, we explored the potential use of the nuclear replicating GmDNV as a vector for the transient expression of foreign genes in insect cells. Several properties of densoviruses make them potential vectors. Like all parvoviruses they are environmentally stable, and their small genome size (5-6 kb) makes them amenable to genetic manipulations. The regulatory proteins have multiple functions in the parvoviral life cycle. They are required for viral DNA replication and are required in trans for efficient expression of the structural promoter. They also regulate their own expression and they were found capable of inhibiting the activity of heterologous (viral and cellular) promoters. If similar functions are carried out by the DNV, NS genes can be utilized in the design of the viral vectors, shownin Figure 6. In addition, the structural similarity to AAV, a human parvovirus shown to integrate at a specific site in chromosome 19 of the human chromosome , makes the study of possible DNV integration challenging. Indeed, data suggesting GmDNV and JcDNV integration was presented recently [26,27]. Like all viral insecticides, the larvacidal activity of GmDNV is slow but the killing rate varies sharply with the differentiation stage of the larvae. The most rapid rate was obtained with 3rd instar larvae, and was at least twofold less with prepupae (Fig. 5). Transcription activity is also highest in 3rd and 4th instar larvae (Fig. 4), suggesting that the larvacidal activity is a consequence of GmDNV replication or gene expression. However, the correlation between larvacidal activity and gene expression is not direct. For example, 4th and 5th instar larvae have similar susceptibility to killing by the virus (Fig. 3), yet the most pronounced difference in mRNA content is between 4th and 5th instars (Fig. 4).It is possible that killing is a consequence of virus replication in specific insect tissues, rather than of the overall levels of gene expression in the entire insect. GmDNV also inhibits pupation of prepupae, and as shown in Figure 354 Tal and Attathom 5, the efficiency of this activity largely depends on the method used to administer the virus. In most DNA viruses, gene expression is divided into clearly separated early and late phases, which occur before and after viral DNA replication, respectively. In mammalian parvoviruses, the early and late genes are expressed in a temporal manner  but the activities of the two groups of genes are largely overlapping and they are not separated by DNA synthesis. We speculate that the lack of such phasing is a consequence of the partially overlapping organization of the regulatory and structural transcription units. It is not clear yet if this time difference seen in Figure 2 reflects true early/late phasing. However, the novel organization of the GmDNV genome offers us a tool to test this hypothesis. These studies, now in progress, are facilitated by the finding that viral DNA is amplified efficiently in cell lines. Viruses have been used for biological control purposes for two decades, but like most biological insecticides, they are slow to act. There is an increasing interest in improving the killing effect of biological control agents which have been genetically engineered to express foreign genes lethal to insects. These attempts, which were done mainly in baculoviruses, included toxins [28-301, hormones  and enzymes . As an approach to overcome the slow killing rate of the virus, we have initiated a long-term study aimed at the construction of a GmDNV with enhanced killing potential. The two prototype vectors constructed are shown capable of expressing transiently the bacterial CAT gene in live larvae, albeit still at low levels (Figs. 6, 7). As a safeguard, these vectors are missing two structural elements which are required for virus production. The inverted terminal repeats, which contain the viral origin of replication, are missing from the constructs. In addition, the structural genes of GmDNV were removed and substituted by the foreign gene. These modifications render these vectors completely deficient for the production and release of virus particles into the environment. LITERATURE CITED 1. Berns KI: Parvovirus replication. Microbiol Rev 54, 316 (1990). 2. Berns KI, Labow MA: Parvovirus gene regulation. J Gen Virol68, 601 (1987). 3. 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