Characterization of a Single-Nucleocapsid Nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia

Journal of Invertebrate Pathology 75, 279 –287 (2000) doi:10.1006/jipa.2000.4933, available online at http://www.idealibrary.com on

Characterization of a Single-Nucleocapsid Nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia Xiao-Wen Cheng 1 and Gerald R. Carner Department of Entomology, 113 Long Hall, Clemson University, Clemson, South Carolina 29634-0365 Received August 16, 1999; accepted March 8, 2000

A single-nucleocapsid nucleopolyhedrovirus (NPV) isolated from Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1.22 ␮m (range ⴝ 0.803–1.931 ␮m). The length of the virion with an envelope averaged 0.29 and 0.23 ␮m without an envelope. ThorNPV was propagated in Pseudoplusia includens (Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV produced in T. orichalcea and P. includens. Polyhedrin amino acid sequence analysis revealed that ThorNPV belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV against third instar P. includens was low, with a LD 50 value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions embedded, which might explain the low virulence against P. includens. Differences in virion occlusion rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD 50 value to P. includens against Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens, P. includens was the only species infected. The virus infected primarily the fat body, tracheal epithelium, and hypodermis. The genomic size of the ThorNPV is 135 kb. © 2000 Academic Press Key Words: Thysanoplusia orichalcea; Pseudoplusia includens; NPV; bioassay; host range; histopathology; morphology; DNA restriction profile; genomic size; Indonesia.

MATERIALS AND METHODS

1. Insect Rearing

INTRODUCTION

Nucleopolyhedroviruses (NPVs) in the family Baculoviridae are common natural microbial control agents 1

that have been used in a number of integrated pest management programs (Podgwaite, 1985; Shepard and Shepard, 1997). NPVs have been the virus of choice for insect control because of their stability and ease of handling. Although 488 isolates of NPVs have been reported (Murphy et al., 1995), relatively few have been developed as microbial insecticides, and efforts are still underway to locate improved strains and species. An NPV was isolated from loopers, Thysanoplusia orichalcea (Lepidoptera:Noctuidae), collected from carrots in West Java, Indonesia, in 1992. Initial observations by light microscopy revealed that the occlusion bodies (OBs) of the virus were quite small and had an unusual tetrahedral shape. The virus was brought to Clemson University for further characterization and for testing against local species of loopers. In initial tests, the crude preparation of the virus showed activity against soybean looper, Pseudoplusia includens (Walker), and cabbage looper, Trichoplusia ni (Hubner). However, this 1992 shipment was subsequently found to contain a mixture of viruses including two NPVs (one is tetrahedral and one is polyhedral in shape) and a cytoplasmic polyhedrosis virus (CPV). A second shipment of NPV-infected T. orichalcea in 1996 contained only the tetrahedral form. When the tetrahedral virus was separated and purified, it showed no activity against T. ni. In this paper, the tetrahedral ThorNPV is characterized as to its morphology, biological activity, histopathology, and molecular biology.

Present address: Laboratory for Molecular Virology, Great Lakes Forestry Center, 1219 Queen St. E., Sault Ste. Marie, ON P6A 5M7 Canada.

P. includens were from a laboratory colony established using eggs obtained from the USDA-ARS, Southern Insect Management Laboratory, Stoneville, Mississippi. All other insects used in the host range test were colonies maintained in our insect rearing facility. Larvae of all insects species were reared individually on pinto bean-based artificial diet (Burton,

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1969) at 27 ⫾ 1°C, 75% relative humidity, and 16:8 h (L:D) photoperiod. Fifty pupa were randomly selected and transferred into a 4-liter glass jar. Emerged adults were fed with 10% honey water. A 10-cm-wide and 25-cm-long paper towel strip was hung in the jar for egg deposition. Eggs on the paper towel strip were transferred into another clean glass container for hatching. Neonates were transferred individually onto the surface of the diet in 31-ml (one ounce) artificial diet cups for development to the pupal stage. 2. Propagation and Purification of ThorNPV Infected T. orichalcea larvae were collected in 1996 from carrots in Indonesia and stored at ⫺20°C until they were transported to the United States. The larvae were macerated in a glass homogenizer with distilled water, and the homogenate was filtered through four layers of cheesecloth. This crude NPV suspension was used to prepare an inoculum (1 ⫻ 10 7 OBs/ml) that was spread on the surface of the diet (0.07 ml/cup) on which third instar soybean loopers were feeding. Infected larvae showing virus infection symptoms were collected and stored at ⫺20°C. OBs from infected larvae were purified by differential and sucrose gradient centrifugation using the method of Cheng et al., (1990). The purified OB pellet was suspended in 0.5 ml of distilled water and stored at ⫺20°C. Purification of virions followed the methods of Harrap and Longworth (1974) and Harrap et al., (1977). Briefly, virions were released from OBs by treatment with 0.1 M sodium carbonate (pH 10.5) and gentle agitation at 37°C until the suspension was relatively transparent and colored light brown. These virions were purified by centrifugation on a linear sucrose gradient (25–50%) at 80,000g for 1 h at 4°C. This gradient was made by freezing 30 ml of 37.5% sucrose (w/v) in the centrifuge tube and thawing at 4°C for 15 h. The virion band was collected with a pasteur pipet, diluted with distilled water, and pelleted at 46,000g for 1 h at 4°C. The purified virion pellet was stored at ⫺20°C. 3. DNA Analysis The DNA from purified virions of ThorNPV was isolated using the resin-based Qiagen Genomic DNA Isolation Kit 100/G (Qiagen). Isolation of DNA followed the cell culture protocol of the kit except that a higher DNA yield was obtained by not vortexing to avoid shearing and performing proteinase K digestion overnight at 37°C. The concentration of isolated DNA was determined by measuring the absorbance at 260 nm and the purity was estimated by calculating the 260: 280 nm absorbance ratio. Autographa californica NPV, AcMNPV (AcUW1-LacZ, a clone from Pharmigen), which was used in the following comparative restric-

tion endonuclease (REN) analysis, was propagated in Sf21 cells and virions were harvested by ultracentrifugation (O’Reilly et al., 1992). DNA from AcMNPV virions was purified as above. REN analysis consisted of digesting 0.5 ␮g of purified DNA from ThorNPV and AcMNPV with HindIII, EcoRI, PstI, and XbaI (Promega or BioLab) in 20-␮l reaction volumes under the conditions recommended by the manufacturer. Digested fragments were separated in a 0.7% agarose gel using ␭-HindIII DNA and 1 kb DNA ladder (GIBCO/BRL) as molecular size markers. DNA from ThorNPV and AcMNPV digested with the same REN was loaded in the gel side by side. The gel was stained with ethidium bromide and photographed under an UV transilluminator with an IS1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA). The accompanying software (V. 2.02) was used to estimate the molecular weight of DNA fragments. Since ThorNPV was propagated in an alternate host other than the original one, confirmation of authenticity was needed. OBs and virions were extracted from a cadaver of T. orichalcea infected by ThorNPV as above. Because the original sample was small in virion yield, phenol– chloroform–isoamylalcohol (25:24:1) DNA extraction was performed following proteinase K digestion as above. To clone the open reading frame (ORF) of the polyhedrin gene for sequencing, the polymerase chain reaction (PCR) method was employed. Basically, a pair of 20-mer primers was chosen from the flanking sequence of the polyhedrin gene ORF of ThorNPV propagated in P. includens (Forward, 5⬘-GCG TCC GTG TAG ATG TAA AG-3⬘; Reverse, 5⬘-CTA TAG CAG CTT CGC GTC TA-3⬘). The forward and reverse primers were located at ⫺208 and ⫹852 nt positions where the A in the translation initiation codon ATG of the polyhedrin gene was set as position 1 (Cheng et al., 1998). Viral DNA from ThorNPV propagated in P. includens was used as a control template in the PCR reaction. Standard PCR protocol was followed. The PCR product was gel purified and ligated into pGEM-T vector (Promega) before transforming DH5␣ competent cells by the electroporation method (Sambrook et al., 1989). Positive clones were selected on LB plates with ampicilin/X-gal/IPTG. Plasmid DNA was purified from three clones carrying the ThorNPV polyhedrin ORF insert. Sequencing of the insert was performed using dye-labeled ALF M13 universal and reverse primers in a Cy5 AutoRead Sequencing Kit with an ALFexpress DNA Analysis System (Pharmacia Biotech). The sequence was processed by a biocomputing software, LASERGENE (DNASTAR, Inc.). This sequence was searched in GenBank (NCBI) using the BLAST search engine. To establish the phylogenetic relationship of this virus to other members in the baculovirus family, the ThorNPV polyhedrin amino acid sequence was aligned with all the homologous polyhedrin/granulin protein

CHARACTERIZATION OF ThorNPV

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FIG. 1. Comparative restriction enzyme digestion profile of ThorNPV and AcMNPV DNA using the restriction endonucleases HindIII, EcoRI, PstI, and XbaI. One-kilobase DNA ladder and ␭-HindIII marker were used as molecular size markers. Each visible band was assigned one or more letters depending on the number of fragments in each band.

hits from the BLAST searches in GenBank using the CLUSTAL method with LASERGENE. The closely related NPVs plus AcMNPV were used for phylogenetic analysis by LASERGENE and PAUP 3.0r (Written by D. L. Swofford). 4. Bioassay Second instar P. includens usually climbed to the paper lids of the diet cups and were transferred individually using a camel hair brush to 1.5-ml Eppendorf tubes with a 1-mm hole punched on the lid of each tube. These larvae were starved overnight, and by the next day they were in early third instar. Fresh tomato leaf disks (4 mm in diameter) were soaked in 0.02% Triton X-100 for 5 min and were washed three times in distilled water. Excess water was removed by blotting with a paper towel. Each disk was then treated with 1 ␮l of ThorNPV suspension at one of five concentrations: 4 ⫻ 10 5, 4 ⫻ 10 6, 4 ⫻ 10 7, 4 ⫻ 10 8, and 4 ⫻ 10 9 OBs/ml.

For the control, leaf disks were treated with 1 ␮l of distilled water. After the leaf disks had air-dried, they were placed individually into the Eppendorf tubes containing the starved soybean looper larvae. One hundred loopers were used to test each concentration, with 4 replications of 25 larvae each. The Eppendorf tubes containing loopers and leaf disks were put into sealed plastic bags with a piece of damp towel in each bag. Two to three holes (2 mm) were punched in the plastic bags to prevent condensation. After approximately 24 h, most loopers had consumed the leaf disks; they were then transferred individually into 31-ml (one ounce) artificial diet cups. Loopers that had not completely consumed the leaf disks were discarded. The bioassay was conducted in an incubation chamber where environmental conditions were the same as in the rearing room. Mortality of loopers was checked and recorded daily until adults emerged from pupae that had not died.

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The dose–mortality data were analyzed with a computer program, POLO-PC (Le Ora Software, Inc., Berkeley, CA), based on the probit analysis method described by Finney (1971).

TABLE 1 Size of ThorNPV DNA Fragments Generated by Digestion with the Restriction Enzymes HindIII, EcoRI, PstI, and XbaI Size (kb) Fragment

HindIII

EcoRI

PstI

XbaI

17.3 14.2 14.2 12.2 8.7 7.1 7.1 6.0 5.0 4.7 4.2 4.2 3.9 3.7 3.4 3.2 3.0 2.9 1.9 1.6 1.6 1.1 1.1 0.8 0.6

23.1 13.8 9.9 9.3 7.5 7.5 6.9 6.9 6.2 4.7 4.7 4.5 4.0 3.8 3.2 3.2 2.8 2.7 2.5 2.3 2.1 1.8 0.9 0.8 0.4

18.1 18.1 11.2 7.1 7.1 6.2 5.9 5.5 5.3 5.0 4.3 4.3 4.3 3.9 3.8 3.7 3.5 2.6 2.4 2.4 1.8 1.8 1.8 1.6 1.3

17.3 12.6 10.7 8.1 7.8 7.5 6.6 6.6 6.3 5.0 5.0 5.0 4.8 4.0 4.0 3.9 3.8 3.7 2.8 2.3 1.8 1.6 1.4 1.3 0.9

Total 134.3 Average ⫾ SEM 134.8 ⫾ 0.4

135.5

5. Host Range Test Purified ThorNPV OB suspension was used to prepare an inoculum (1 ⫻ 10 7 OBs/ml) that was spread on the surface of the diet in 31-ml diet cups (730 OBs/ mm 2). Thirty to eighty third instar larvae of Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens were transferred individually to the viruscontaminated diet. Development of larvae was checked daily. 6. Electron Microscopy For scanning electron microscopy (SEM), the purified NPV pellet was suspended in 5 ␮l of distilled water. One microliter of the viral suspension was spread onto a 1 ⫻ 1-cm glass slide, dehydrated in an oven at 37°C for 30 min, and coated with gold. The coated sample was observed under the SEM (JEOL 848) and pictures were taken and saved on an optical disk for measurement of virus structures using the PGT IMIX software in a Sun workstation. For transmission electron microscopy (TEM), 30 third instar soybean loopers were starved for 6 h. Loopers were held individually in 1.5-ml Eppendorf tubes with a hole in the lid and allowed to feed on a 4-mmdiameter tomato leaf disc treated with 1 ␮l of virus suspension. When the loopers consumed the leaf disc (about 5 h), they were transferred into artificial diet cups. Every 12 h postinoculation (p.i.), a looper was taken out and dissected to collect the fat body. At 7 days p.i., the midgut, tracheae, hypodermis, silk glands, Malpighian tubules, and testes were also collected. Tissue samples were immersed in 2% phosphate-buffered glutaraldehyde for 2 h. Processing of samples for TEM followed the methods of Hamm and Styer (1985). The polymerized samples were sectioned with a diamond knife on a microtome (Reichert-Jung Ultracut-E), poststained with uranyl acetate and lead citrate (Venabel and Coggleshall, 1965), and examined under TEM (Hitachi H600 AB).

A B C D E F G H I J K L M N O P Q R S T U V W X Y

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135.6

RESULTS

A single band formed in the sucrose gradient centrifugation of dissolved OBs. This indicated that the virions were all of the same size. The virions were contained at about the 35% (w/v) region of the sucrose gradient. The freeze-and-thaw method for formation of the linear sucrose gradient provided a very efficient and simple way to purify the virus particles. Comparative REN analysis of the ThorNPV genomic DNA with AcMNPV is shown in Fig. 1. There were 26 HindIII, 25 EcoRI, 26 PstI, and 26 XbaI REN recognition sites in the ThorNPV genome. The genome size of the ThorNPV was estimated to be 134.8 ⫾ 0.4 kb (Table 1). REN profiles also showed that the ThorNPV

FIG. 2. Alignment of polyhedrin amino acid sequences from NPVs that are closely related to ThorNPV. Residues in boxes represent those that differ from the consensus. Dots in the consensus indicate that not all residues match. Autographa californica NPV, AcMNPV (Hooft van Iddekinge et al., 1983); Leucania seperata NPV, LeseNPV (GenBank Access No. U30302); Mamestra brassicae NPV, MbMNPV (Cameron and Possee, 1989); Mamestra configurata NPV, MacoNPV (Li et al., 1997); Panolis flammea NPV, PaflNPV (Oakey et al., 1989); Buzura suppressaria single-nucleocapsid NPV, BusuNPV (Hu et al., 1993); Orgyia pseudotsugata single-nucleocapsid NPV, OpSNPV (Leisy et al., 1986); Ectropis obliqua single-nucleocapsid NPV, EcobNPV (U95014); Trichoplusia ni single-nucleocapsid NPV, TrniNPV (Fielding and Davison, 1999); Thysanoplusia orichalcea single-nucleocapsid NPV, ThorNPV (Cheng et al., 1998); Spodoptera exigua NPV SeMNPV (Van Strien et al., 1992); Spodoptera frugipeda NPV SfMNPV (Gonzalez et al., 1989); Spodoptera litura NPV, SpliNPV (X94437).

CHARACTERIZATION OF ThorNPV

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FIG. 4. Accumulated mortality of third instar soybean loopers treated with different dosages of ThorNPV using a leaf disk bioassay. Control, 0 OBs/larva. The vertical line for each dose represents the standard error for the mean, n ⫽ 74.

FIG. 3. The most parsimony tree (unrooted) of some polyhedrin genes from NPVs closely related to ThorNPV after initial alignment of all NPV polyhedrin genes available in GenBank as of January 2000 with Clustal method. The tree was constructed after 100 replicates of bootstrap branch-and-bound search using PAUP 3.0r. I and II are two groups of baculoviruses (Zanotto et al., 1993). Percentages in the parentheses beside the virus name are polyhedrin sequence identities with ThorNPV. Numbers above the lines are the number of changes between the node and the species. Numbers in italics below the line indicate the frequency of that cluster after bootstrap analysis. (CI ⫽ 0.78; RI ⫽ 0.72).

was readily distinguishable from the type species of baculovirus, AcMNPV (Fig. 1). When ThorNPV DNAs from OBs propagated in both T. orichalcea and P. includens were used as templates in the PCR amplification, both PCR reactions produced an expected 1-kb fragment (photo not shown). After the 1-kb polyhedrin PCR product from ThorNPV originating in T. orichalcea was cloned and sequenced, a search of GenBank using BLAST revealed that this ThorNPV polyhedrin gene from virus produced in T. orichalcea was identical to that of ThorNPV recovered from P. includens (Cheng et al., 1998) (Access Number AF019882). Polyhedrin gene sequence alignment showed that ThorNPV was a new member of Group II of the Baculoviridae (Zanotto et al., 1993). Based on polyhedrin protein sequence within Group II, ThorNPV formed a subcluster with other NPVs, with its closest relative being T. ni NPV (TrniNPV) (Fielding and Davison, 1999) (Figs. 2 and 3). The two viruses share 97.6% polyhedrin amino acid sequence identity. There is only a five amino acid difference between the ThorNPV and TrniNPV polyhedrin protein sequence (Fig. 2). About 4000 OBs of ThorNPV per larva were required to produce noticeable mortality in third instar soybean loopers. Concentrations lower than 4000 OBs per larva did not produce mortality (Fig. 4). The LD 10, LD 50, and LD 90 values for the ThorNPV against third instar soy-

bean loopers were 5194, 65,636, and 829,394 OBs/ larva, respectively (Table 2). The equation of the log dose–probit regression line was Y ⫽ 1.1633x ⫺ 5.6040 (x 2 ⫽ 5.2759; df ⫽ 3), the standard error (SE) of the slope was found to be 0.0880, and the t value was 13.2206. The estimated t value (13.2206) greatly exceeded 2.576 (1% significance point for the t distribution with ⬁ df), which implied the probit response is a linear function of log dose in this bioassay. Seven species of Noctuidae, including P. includens, were tested for infection by ThorNPV. Only P. includens was infected by this virus (54.7%), which was determined by examining the dead larvae for large numbers of tetrahedral NPV OBs. However, this NPV was found to be infective against another looper, Chrysodeixis chalcites, in Indonesia (unpublished data). The amount of inoculum used in the host range test was approximately the LD 50 value (65,636 OBs/larva) to P. includens. As seen by SEM, the occlusion shape of the ThorNPV is roughly tetrahedral. The triangular faces were usually not uniform and bulbous protrusions were observed on most OBs. Some virions were not completely embedded and “pits” were left on the OBs when virions were lost during the preparation (Fig. 5). The average

TABLE 2 Dosage Mortality Response of Third Instar Soybean Looper, P. includens, to ThorNPV a 95% Confidence limits Entry

Value

Upper

Lower

LD 10 (OBs/larva) LD 50 (OBs/larva) LD 90 (OBs/larva) Slope

5,194 6,5636 829,394 1.163

12,443 135,908 3,510,570

1,216 31,615 347,528

a

LD values in the table were calculated with a computer program, POLO-PC.

CHARACTERIZATION OF ThorNPV

FIG. 5. Scanning electron micrograph (15 kV, ⫻8000) showing the ThorNPV occlusion bodies. Solid arrow indicates “pits” in the OBs where virions were embedded but were lost in the preparation. BS, bulbous surface.

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size of the OBs was 1.37 ⫾ 0.43 ␮m with a range of 0.803 to 1.931 ␮m. Ultrathin sections of the ThorNPV revealed that this was a single-nucleocapsid NPV (Fig. 6A). Among the tissues examined, it was found that ThorNPV replicated mainly in the nuclei of fat body and tracheal epithelium cells. Although hypodermis was not observed under TEM, it was probably also infected because the integument became very fragile in late stages of infection. Virus nucleocapsid assembly occurred in the virogenic stroma in the nuclei of infected cells in tracheal epithelium and fat body (Fig. 6B). The size of the virion with envelope was 0.290 ⫾ 0.06 ⫻ 0.068 ⫾ 0.004 ␮m, and the size without the envelope was 0.230 ⫾ .06 ⫻ 0.035 ⫾ .004 ␮m. Abnormal OBs were found with few or no virions and many virions were not occluded in the later stages of infection (Fig.

FIG. 6. Transmission electron micrographs of soybean looper fat body cells infected with ThorNPV. (A) One hundred eighty hours postinfection (p.i.). Arrow indicates a normal occlusion body with virions embedded. (B) Seventy-two hours p.i. showing the virogenic stroma (VS), nucleocapsids (NC) without envelopes, virions (V) and polyhedrin membrane (PM) scattered in the nucleus of the cell. (C) One hundred eight hours p.i. Arrowhead indicates occlusion body without virions embedded. Solid arrow points to virions not occluded. (D) OBs in two adjacent cells in the tracheal hypodermis. One cell has normal OBs with embedded virions (a) and the other has abnormal OBs with few or no virions embedded (b).

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6C). In some cells, clumps of polyhedrin could be seen scattered through the nucleus (Fig. 6B). This was observed in both fat body and tracheal epithelium tissues. In examining clusters of infected cells, we would often observe one cell with fully formed normal OBs and an adjacent cell would contain abnormal OBs with no embedded virions (Fig. 6D). DISCUSSION

The genomic size is an important characteristic of a baculovirus at the molecular level. It has been reported that the genome of baculoviruses is highly complex and occurs as circular, supercoiled, dsDNA of 88 to 160 kb (Blissard and Rohrmann, 1990). The genome size of ThorNPV (134.8 kb) falls within this reported range. Use of REN digestion for the estimation of viral genome size in this research resulted in a very small variation in size estimates (Table 1). There were concerns that the ThorNPV produced in P. includens was not authentic. However, a comparison of polyhedrin sequences revealed that virus recovered from P. includens was the same as that recovered from the original host. This was further confirmed by a side by side comparison of HindIII DNA profile of ThorNPV produced in the original host, T. orichalcea, and the alternate host, P. includens, in which no noticeable difference was observed (photo not shown). The ThorNPV was found to be closely related to TrniNPV, based on high polyhedrin sequence identity (97.6%). Both are single-nucleocapsid NPVs, but they are not identical (Figs. 2 and 3). They differ not only in polyhedrin sequence but also in the shape of the OB, with TrniNPV forming a polyhedron and ThorNPV a tetrahedron (Fielding and Davison, 1999; Cheng et al., 1998). Furthermore, ThorNPV is not able to infect T. ni larvae. The tetrahedral shape of ThorNPV did not change when it was produced in the alternate host, P. includens. Similar results were also reported by Young and Yearian (1983) who observed that the tetrahedral shape of Rachiplusia nu NPV (RanuNPV) did not change when it was passed through an alternate host, Rachiplusia ou. The ThorNPV showed very low virulence to third instar P. includens (Table 2), which may be explained by the fact that P. includens is not the native host for ThorNPV. Livingston et al. (1980) reported an NPV from P. includens and found this P. includens singlenucleocapsid NPV was very virulent to third instar soybean loopers (LC 50 ⫽ 5.7 PIB/mm 2). Tetrahedral-shaped NPVs are rarely isolated, and this is one of the reasons for our interest in studying ThorNPV. There are a few reports of other NPVs with a similar shape. Young and Yearian (1983) reported an NPV from Rachiplusia nu with a tetrahedral shape and Lymantria monacha (Linn.) and Hyphantria cunea (Drury) also have NPVs with tetrahedral OBs (Smith,

1976). However, no detailed molecular studies have been conducted with these other NPVs. The size of the ThorNPV polyhedra is at the lower end of the size range (0.5–15 ␮m) reported for NPVs (Tanada and Kaya, 1993). The tetrahedral-shaped RanuNPV reported by Young and Yearian (1983) showed a similar size of 1.16 ␮m with a range of 0.72 to 1.66 ␮m. Abnormal OBs were found with few or no virions or with the virions not completely occluded (Figs. 5 and 6A). Similar abnormalities have also been found in S. exigua infected with the S. frugiperda MNPV (Hamm and Styer, 1985) and Lambdina fiscellaria somniaria NPV in a cell line of Malacosoma distria hemocytes (Sohi and Cunningham, 1972). These reports suggest that malformation of OBs may be due to replication in an alternate host rather than in the native host. However, an examination of the inoculum of ThorNPV from its original host also showed some OBs with few or no virions (photo not shown). A difference in virion occlusion was observed in adjacent cells in the same tissue (Fig. 6D). If we assume that the two neighboring cells present the same conditions for virus replication, then the virion occlusion difference may reside in the virus itself. Most NPVs that are isolated from the field and propagated in laboratory hosts are a mixture of different variants of the same virus. Some of the cells may have been infected by variants with a malfunctioned gene(s) that controls virion occlusion. Evidence of this was presented by Slavicek et al. (1998) who identified a novel Lymantria dispar NPV mutant (PFM-1) that had very few viral nucleocapsids in the OBs. By using marker rescue studies, they gave proof of the existence of genes other than polyhedrin and 25 K FP that directly impact virion occlusion. This occurrence of OBs with few or no virions may contribute in part to the low virulence of this virus to the soybean looper, since OB counts are used in dosage determination. Thus, it would be important for commercial producers to check abnormality rates when mass-producing baculoviruses as viral insecticides. In conclusion, based on the polyhedrin gene sequence searches in GenBank, ThorNPV is a new member of the family of Baculoviridae. Its potential as a candidate for microbial control of local looper species is very questionable. However, the embedding abnormality of ThorNPV in vivo may be useful in studying the factor(s) that govern this abnormality. ACKNOWLEDGMENTS The authors thank Drs. Eleanor and Merle Shepard for collection of NPV sample in Indonesia. We also thank JoAn S. Hudson and Ai-Teh Zhang of the Clemson Electron Microscope facility for assistance in the operation of both TEM and SEM and Dr. Basil Arif for help in completing the manuscript. Financial support was provided by HATCH Project 1614 and by a USAID Project, Integrated Pest Management Research, Development and Training Activities for

CHARACTERIZATION OF ThorNPV Palawija Crops in Indonesia. This paper is Technical Contribution 4398 of the South Carolina Agricultural Experiment Station, Clemson University. REFERENCES Blissard, G. W., and Rohrmann, G. F. 1990. Baculovirus diversity and molecular biology. Annu. Rev. Entomol. 35, 127–155. Burton, R. L. 1969. “Mass Rearing the Corn Earworm in the Laboratory.” USDA Agricultural Research Series ARS-134. Cameron, I. R., and Possee, R. D. 1989. Conservation of polyhedrin gene promoter function between Autographa californica and Mamestra brassicae nuclear polyhedrosis viruses. Virus Res. 12, 183– 199. Cheng, X.-W., Aguda, R. M., and Shepard, B. M. 1990. A nuclear polyhedrosis virus from rice skipper. Int. Rice Res. Inst. Newsl. 15(1), 33–34. Cheng, X.-W., Carner, G. R., and Fescemyer, H. W. 1998. Polyhedrin sequence determines the tetrahedral shape of occlusion bodies in Thysanoplusia orichalcea single-nucleocapsid nucleopolyhedrovirus. J. Gen. Virol. 79, 2549 –2556. Fielding, B. C., and Davison, S. 1999. The characterization and phylogenetic relationship of the Trichoplusia ni single capsid nuclear polyhedrosis virus polyhedrin gene. Virus Genes 19(1), 67– 72. Finney, D. J. 1971. “Probit Analysis,” 3rd ed. Cambridge Univ. Press, Cambridge. Gonzalez, M. A., Smith, G. E., and Summers, M. D. 1989. Insertion of the SfMNPV polyhedrin gene into an AcMNPV polyhedrin deletion mutant during viral infection. Virology 170, 160 –175. Hamm, J. J., and Styer, E. L. 1985. Comparative pathology of isolates of Spodoptera frugiperda nuclear polyhedrosis virus in S. frugiperda and S. exigua. J. Gen. Virol. 66, 1249 –1261. Harrap, K. A., and Longworth, J. F. 1974. An evaluation of purification methods for baculoviruses. J. Invertebr. Pathol. 24, 55– 62. Harrap, K. A., Payne, C. C., and Robertson, J. S. 1977. The properties of three baculoviruses from closely related hosts. Virology 79, 14 –31. Hooft van Iddekinge, B. J. L., Smith, G. E., and Summers, M. D. 1983. Nucleotide sequence of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. Virology 131, 561–565. Hu, Z. H., Liu, M. F., Jin, F., Wang, Z. X., Liu, X. Y., Li, M. J., Liang, B. F., and Xie, T. E. 1993. Nucleotide sequence of the Buzura suppressaria single nucleocapsid nuclear polyhedrosis virus polyhedrin gene. J. Gen. Virol. 74, 1617–1620. Leisy, D., Nesson, M., Pearson, M., Rohrmann, G., and Beaudreau, G. 1986. Location and nucleotide sequence of the Orgyia pseudotsugata single nucleocapsid nuclear polyhedrosis virus polyhedrin gene. J. Gen. Virol. 67, 1073–1079. Li, S., Erlandson, M., Moody, D., and Gillott, C. 1997. A physical map of the Mamestra configurata nucleopolyhedrovirus genome and

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sequence analysis of the polyhedrin gene. J. Gen. Virol. 78, 265– 271. Livingston, J. M., McLeod, P. J., Yearian, W. C., and Young, S. Y. 1980. Laboratory and field evaluation of a nuclear polyhedrosis virus of the soybean looper, Pseudoplusia includens. J. Ga. Entomol. Soc. 15, 194 –199. Murphy, F. A., Fauquet, C. M., Bishop, D. H. L., Ghabrial, S. A., Jarvis, A. W., Martelli, G. P., Mayo, M. A., and Summers, M. D. 1995. “Virus taxonomy: Sixth report of the International Committee on Taxonomy of Viruses.” Springer-Verlag, Berlin/New York. Oakey, R., Cameron, I. R., Davis, B., Davis, E., and Possee, R. D. 1989. Analysis of transcription initiation in the Panolis flammea nuclear polyhedrosis virus polyhedrin gene. J. Gen. Virol. 70, 769 –775. O’Reilly, D. R., Miller, L. K., and Luckow, V. A. 1992. “Baculovirus Expression Vectors—A Laboratory Manual.” Oxford Univ. Press, New York. Podgwaite, J. D. 1985. Strategies for field use of baculoviruses. In “Viral Insecticides for Biological Control” (K. Maramorosch and K. E. Sherman, Eds.), pp. 775–797. Academic Press, Orlando. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Shepard, B. M., and Shepard, E. F. 1997. “Integrated pest management research; management and training activities for palawija crops in Indonesia.” United States Agency for International Development, Final Report, Washington, DC. Slavicek, J. M., Mercer, M. J., Pohlman, D., Kelly, M. E., and Bischoff, D. S. 1998. Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion. J. Invertebr. Pathol. 72, 28 –37. Smith, K. M. 1976. “Virus–Insect Relationships.” Longman, New York. Sohi, S. S., and Cunningham, J. C. 1972. Replication of a nuclear polyhedrosis virus in serially transferred insect haemocyte cultures. J. Invertebr. Pathol. 19, 51– 61. Tanada, Y., and Kaya, H. K. 1993. “Insect Pathology.” Academic Press, San Diego. Van Strien, E. A., Zuidema, D., Goldbach, R. W., and Vlak, J. M. 1992. Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus. J. Gen. Virol. 73, 2813–2821. Venabel, J. H., and Coggleshall, R. 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407– 408. Young, S. Y., and Yearian, W. C. 1983. Pathology of a nuclear polyhedrosis virus of Rachiplusia nu in Rachiplusia ou (Lepidoptera: Noctuidae). J. Invertebr. Pathol. 42, 410 – 412. Zanotto, P. M., Kessing, B. D., and Maruniak, J. E. 1993. Phylogenetic interrelationship among baculoviruses: Evolutionary rates and host associations. J. Invertebr. Pathol. 62, 147–164.