Journal of Invertebrate Pathology 88 (2005) 126–135

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Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.)

(Lepidoptera: Noctuidae)�

Xiao-Wen Chenga,b,d,¤, Gerald R. Carnerb, Martin Langec, Johannes A. Jehlec, Basil M. Arif d

a Department of Microbiology, 32 Pearson Hall, Miami University, Oxford, OH 45056, USAb Department of Entomology, 113 Long Hall, Clemson University, Clemson, SC 29634, USA

c Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate, 67435 Neustadt and der Weinstraße, Germany

d Laboratory for Molecular Virology, Great Lakes Forestry Center, 1219 Queen St. E. Sault Ste Marie, Ont., Canada P6A 2E5

Received 20 August 2004; accepted 24 December 2004

Abstract

A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixedinfected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of Thor-MNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band proWles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from diVerent virionsample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 valuesof 17 and 242 OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera fru-giperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as thefat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA trans-fection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame(ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phyloge-netic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and isrelated but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster. 2005 Elsevier Inc. All rights reserved.

Keywords: Thysanoplusia orichalcea MNPV; Host range; Virus infectivity; Pseudoplusia includens; Trichoplusia ni; DNA restriction proWles;Biological control; Phylogenetic analysis

1. Introduction

By virtue of their speciWcity, virulence and safety fornon-target species, insect viruses, particularly baculovi-

� The sequences reported in this paper has been deposited in Gen-Bank/NCBI with the Accession Nos. AF169480, AY706587,AY706652, and AY706719.

¤ Corresponding author. Fax: +1 513 529 2431.E-mail address: Chengx@muohio.edu (X.-W. Cheng).

0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2004.12.003

ruses, have become promising alternative candidates tochemicals for controlling agricultural and forest insectpests (Podgwaite, 1985). Several baculoviruses have beenin general use around the world for the control of insectpest populations (Bedford, 1981; Cunningham and Ent-wistle, 1981; Moscardi, 1999; Shepard and Shepard, 1997).

Larvae of a looper species, Thysanoplusia orichalcea,infected with a nucleopolyhedrovirus (NPV) were col-lected from carrots in 1992 in West Java, Indonesia and

X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135 127

were brought to Clemson University, South Carolina,USA to test for infectivity against the soybean looper,Pseudoplusia includens. Two NPVs were isolated, one witha tetrahedral occlusion body (OB) and the other with apolyhedral OB. Analyses of the tetrahedral-shaped NPVrevealed a single nucleocapsid NPV belonging to GroupII NPVs (Cheng and Carner, 2000; Cheng et al., 1998;Jehle, 2004). In this paper, we report the characterizationof the second virus with polyhedral OBs.

2. Materials and methods

2.1. Insects

Pseudoplusia includens eggs were obtained from theUSDA Southern Insect Management Laboratory inStoneville, Mississippi, and other insects used in thisstudy were colonies maintained on pinto bean artiWcialdiet in 31-ml (one ounce) plastic cups with paper lids inour insect rearing facility (Burton, 1969). The insect cul-tures were maintained according to the methods ofCheng and Carner (2000).

2.2. Isolation of ThorMNPV

The ThorMNPV was separated from the ThorSNPVby inoculating Trichoplusia ni larvae with the originalmixture of viruses since ThorSNPV was not able toreplicate in T. ni larvae (Cheng and Carner, 2000). Sev-enty microliters of semi-puriWed original NPV inocu-lum (»107 OBs/ml) were spread on the surface of thediet of third instar T. ni larvae and larvae were incu-bated at 27 °C. Infected larvae showing swollen bodyand pale color were collected in a container and storedat ¡20 °C. Propagation of ThorMNPV was carried outin either T. ni or P. includens. In the following studies,OBs from P. includens were used unless otherwiseindicated.

OBs from ThorMNPV infected larvae of P. includensand T. ni were puriWed separately by diVerential andsucrose gradient centrifugation (Cheng et al., 1990). ThepuriWed OB pellet was suspended in 0.5 ml of distilledwater and quantiWed using a hemocytometer. The viruspreparation was used as a stock for DNA analyses andbioassays.

Virions were puriWed according to the methods ofHarrap and Longworth (1974) and Cheng and Carner(2000). The puriWed ThorMNPV virions were used forDNA extraction or stored at ¡20 °C.

2.3. DNA analysis

DNA was extracted from puriWed virions of Thor-MNPV either produced in P. includens or T. ni larvaeby the phenol method. To check if the ThorMNPV was

contaminated with ThorSNPV, the DNA samples wereused as templates in PCRs where the primers used inThorSNPV polyhedrin analysis were used for theampliWcation (Cheng and Carner, 2000; Cheng et al.,1998). ThorSNPV DNA was used as a positivecontrol.

Restriction endonuclease (REN) analysis was car-ried out by digesting 500 or 200 ng of puriWed Thor-MNPV DNA with HindIII, EcoRI, PstI, and XbaI(Promega or Gibco-BRL) in 20 �l reaction volumes.Digested fragments were fractioned on 0.7 and 0.4%agarose gels, respectively. The gels were stained withethidium bromide and photographed with an IS-1000Digital Imaging System (Alpha Innotech, San Lean-dro, CA). The accompanying software (V. 2.02) wasused to estimate the molecular weight of DNAfragments.

To locate DNA fragments containing the polyhedringene of ThorMNPV, 500 ng of viral DNA were cleavedwith HindIII, EcoRI, PstI, and XbaI and separated on a0.7% agarose gel. The DNA fragments were transferredto a nylon membrane (Hybond-N+, Amersham) by usingthe standard alkaline method (Chomczynski, 1992;Southern, 1975). A 2.5 kb SalI fragment containing thepolyhedrin gene from Orgyia pseudotsugata MNPV(OpMNPV) cloned in pBluescript vector was kindly pro-vided by G.F. Rohrmann (Oregon State University). A1.85 kb fragment containing most of the polyhedrin geneof OpMNPV was obtained by digestion of the plasmidwith XbaI followed by fractionation on a 0.7% agarosegel (Rohrmann et al., 1982). The 1.85 kb fragment wasgel-puriWed and labeled using the digoxygenin randomprimer extension method according to the manufac-turer’s protocol for the Genius I kit (BoehringerMannheim).

DNA–DNA hybridization was performed at 50 °Cwith 20 ng/ml of the digoxigenin-labeled probe for 16 h.The blot was washed twice for 5 min in ample 2£ SSCcontaining 0.1% SDS at 22 °C, and twice for 15 min in0.1£ SSC containing 0.1% SDS at 62 °C. Chromogenicdetection (Genius I) was used to visualize the hybridizedprobe. Restriction fragments containing the polyhedringene of ThorMNPV were identiWed from the hybridiza-tion patterns.

To clone and sequence the polyhedrin gene of Thor-MNPV, viral DNA was digested with REN and sepa-rated on a 0.7% agarose gel. The fragments containingthe putative polyhedrin gene were cloned into a plasmid(pBluescript II SK+, Stratagene) and ampliWed in Esche-richia coli (DH5�). Putative plasmids containing thepolyhedrin gene were digested with the appropriateenzymes and fractionated on a 0.7% agarose gel followedby Southern analysis to screen for fragments hybridizingto the OpMNPV-polyhedrin gene probe. Hybridizingfragments were puriWed and cloned followed by DNAsequencing.

128 X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135

An ABI Prism Dye Terminator Cycle SequencingReady Reaction Kit (Perkin-Elmer, Norwalk, Connecti-cut) was used for all sequencing performed. M13forward and reverse primers were used to producesequences of about 400 nucleotides of both the Wrst andsecond strands of a sub-cloned restriction fragment.Comparison of this initial sequence information withnucleotide sequences for NPV polyhedrin genes wasconducted by searching in GenBank (NCBI). The initialpartial sequence of the ThorMNPV polyhedrin gene wasconWrmed and used to design primers needed tocomplete the sequencing of the polyhedrin gene andthe Xanking region on both strands. Analyses of thepolyhedrin gene sequence were conducted with a DNA

analysis program, LASERGENE (DNASTAR).

2.4. Phylogenetic analyses

For phylogenetic analyses, partial sequences of thelef-8, lef-9, and polh were ampliWed by using the degen-erate primer method described by Lange et al. (2004).Both strands of the PCR fragments were directlysequenced using universal primers. The deduced aminoacid sequence of partial lef-8, lef-9, and polh was con-catenated and aligned with other NPV sequences usingClustal W (Thompson et al., 1994) implemented in theBioedit software (Hall, 1999). Maximum parsimony(MP) phylogenetic trees (1000 bootstrap replicates)were inferred from the amino acid sequence alignmentsusing MEGA, version 2.1 (Kumar et al., 2001). Intro-duced gaps were treated as missing data. A min-miniheuristic search method with a search factor D 2 wasapplied. DayhoV distance corrected neighbor joining(NJ) distance analyses (gamma shape parameter� D 2.25) (Nei and Kumar, 2000) and minimum evolu-tion (ME) analyses (1000 bootstrap replicates) wereperformed using MEGA (Kumar et al., 2001).

2.5. Transfection and infection tests in vitro

Transfection of ThorMNPV viral DNA to two celllines, T. ni High 5 and SF-21, were performed usingMetafectene (Biontex, Germany) mixed with 200 ng ofpuriWed ThorMNPV DNA according to standard pro-cedures (O’Reilly et al., 1992). Transfected cells wereobserved daily for the presence of OBs using aninverted microscope. Budded viruses in media (10 �l)from the well of infected cells were inoculated into25 cm2 Xasks containing monolayers of High 5 or SF-21 cells. Infection was documented with a digital cam-era attached to the microscope.

2.6. In vivo infection experiments

For host range studies, a puriWed ThorMNPV OB sus-pension was used to prepare an inoculum (1 £107 OBs/

ml) that was spread on the surface of the artiWcial diet(730 OBs/mm2). Thirty to 80 third instar larvae of Spo-doptera exigua, S. frugiperda, S. eridania, Anticarsia gem-matalis, Helicoverpa zea, T. ni, and P. includens weretransferred individually to the virus contaminated diet.The development of larvae was checked daily.

Larvae of P. includens and T. ni were used in bioassaysto determine LD50s. Autographa californica nucleopolyhe-drovirus (AcMNPV) was also included in the assayagainst T. ni for comparison with ThorMNPV. A leaf diskassay method used in this experiment was conductedaccording to Cheng and Carner (2000). Forty to 100 thirdinstar larvae were used for each concentration. In the test,30, 150, 300, 1500, and 3000 OBs per larva of ThorMNPVwere used against P. includens. For ThorMNPV againstT. ni, the virus stock was diluted with water to prepareconcentrations of 0.02, 0.2, 2, 20, 200, and 2000 OBs perlarva in the assay. For AcMNPV in T. ni, the virus stockof 2.9£107 OBs/ml was diluted with water to prepare con-centrations of 0.29, 2.9, 29, 290, and 2900 OBs per larva.

The dose-mortality data were analyzed with the com-puter program POLO-PC (Le Ora Software, Berkeley,CA), based on the probit analysis method described byFinney (1971).

2.7. Electron microscopy

PuriWed ThorMNPV OBs were spread onto a 1 £ 1-cm glass slide. The sample was dehydrated in an oven at37 °C for 30 min and coated with gold. The coated sam-ple was observed under a scanning electron microscope(SEM, Joel 848) and photographed.

For transmission electron microscopy (TEM) work, 30third instar P. includens larvae were starved for 6h. Indi-vidual larvae were allowed to feed on a 4 mm diametertomato leaf disc contaminated with 1�l (»500 OBs) ofvirus suspension in a 1.5 ml tube having a hole in its lid.When the loopers consumed the leaf disc (about 5 h), theywere transferred onto artiWcial diet. Every 12h post-inocu-lation (p.i.) a larva was dissected and the fat body tissuewas isolated. At 7 days p.i. other tissues were dissectedincluding midgut, tracheae, hypodermis, silk gland, Malpi-ghian tubules, and testes. Tissue samples were processedfor TEM according to Granados and Lawler (1981).

3. Results

3.1. PuriWcation of ThorMNPV

ThorMNPV was successfully isolated from a mixtureof ThorSNPV and ThorMNPV by passing the mixedinoculum through T. ni larvae. This procedure was basedon the evidence that ThorSNPV could not be transmit-ted to T. ni larvae. In the sucrose gradient centrifugationof virions from OBs produced in T. ni. or P. inlcudens for

X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135 129

DNA extraction, a total of eight discrete virion bandswere obvious. The intensity of each band diVered, withvirion bands 3–6 (counting from top to bottom) brighterthan the rest. This was the case for virions produced inboth insects. The banding pattern appeared to be stablein at least 10 rounds of diVerent virion sample puriWca-tion processes (data not shown).

3.2. DNA analysis of ThorMNPV

To prove the purity of the prepared ThorMNPV sam-ple, PCR ampliWcation using ThorSNPV speciWc prim-

ers (Cheng and Carner, 2000) were performed on theDNA puriWed from ThorMNPV OBs produced in T. nilarvae inoculated with mixed viruses. The negative out-come of this experiment demonstrated that the samplesdid not contain ThorSNPV DNA detectable by PCR(data not shown).

The proWles of HindIII, EcoRI, PstI and XbaI RENdigests of ThorMNPV DNA fractioned on 0.7 and 0.4%agarose gels are shown in Figs. 1A and B. The REN pro-Wles of DNA extracted from ThorMNPV OBs propa-gated in P. includens and T. ni larvae were identical (datanot shown). Side by side comparison of DNA XbaI pro-

Fig. 1. Restriction endonuclease proWles and genomic size estimation of ThorMNPV and comparative XbaI proWle between ThorMNPV andAcMNPV. (A) Restriction fragments resolved in a 0.7% agarose gel. (B) Restriction fragments resolved in a 0.4% agarose gel. Fragment size is in kbp.Number in the parenthesis represents the number of fragments in that band based on densitometer counts. * The fragments containing polyhedringene elucidated from Southern hybridization from Fig. 2 with OpMNPV polyhedrin gene probe.

130 X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135

Wle of ThorMNPV and AcMNPV readily distinguishedthe ThorMNPV from the latter type species of Baculovi-ridae (Fig. 1A). The estimated genome size for the Thor-MNPV is 136 § 0.3 kb (n D 4) (Figs. 1A and B).

Southern hybridization of the digoxygeninlabeledpolyhedrin gene of OpMNPV against digested DNA ofThorMNPV showed speciWc hybridization with the 7.0,6.7, 17.3, and 30.9 kb fragments of HindIII, EcoRI, PstI,and XbaI digests, respectively, indicating that the Thor-MNPV polyhedrin is harbored within these fragments(Fig. 2). The 6.7 kb EcoRI fragment was chosen forcloning, sub-cloning, and identiWcation of the polyhe-drin gene. By Southern hybridization, a 3.5 kb HindIII(one HindIII site from vector) fragment of the 6.7 kbfragment was found containing the polyhedrin gene.This fragment was cloned and subsequently sequencedwith M13 forward and reverse primers and primerwalking. A 1158 bp sequence was obtained, and thiscontained the complete ThorMNPV polyhedrin gene.Sequence alignment with other NPV polyhedrin genesrevealed that the ORF contained 735 nucleotides andencoded 245 amino acids of the putative polyhedrinprotein with an estimated molecular weight of28,705 Da (Fig. 3). The putative transcription start sitewith the TAAG core sequence (Rohrmann, 1986) is

Fig. 2. Southern hybridization analysis to locate polyhedrin gene con-taining restriction fragments of ThorMNPV. ThorMNPV DNA(500 ng) was cleaved with HindIII, EcoRI, PstI, and XbaI, followed by0.7% agarose gel electrophoresis and immobilization of DNA frag-ments to a nylon membrane. A digoxigenin labeled OpMNPV polyhe-drin gene probe was allowed to hybridize to DNA fragments ofThorMNPV on the nylon membrane to detect polyhedrin gene con-taining fragments of ThorMNPV.

located 51 bp upstream of the predicted translationstart codon (ATG) of the polyhedrin ORF. The puta-tive polyadenylation site sequence (AATAAA) islocated immediately at the end of the ORF (Fig. 3).ThorMNPV polyhedrin gene promoter sequence isidentical to that of the AcMNPV (Fig. 3).

Initial analysis of the polyhedrin gene sequence sug-gested that ThorMNPV belongs to Group I NPVs. Todetermine the phylogenetic position of ThorMNPV,partial sequences of the lef-8, lef-9, and polh genes wereampliWed by PCR followed by sequencing of the PCRproducts. Phylogenetic analyses of the deduced concat-enated partial amino acid sequences of lef-8, lef-9, andpolh showed that ThorMNPV belongs to Group INPVs and has a common ancestor with AcMNPV,BmNPV, and RoMNPV (Fig. 4). Based on the distanceto these three viruses, ThorMNPV may be regarded asa diVerent virus isolate.

3.3. Transfection and infection tests in vitro

At three days post-transfection, OBs were observed inthe nuclei of infected High 5 cells but not in the SF-21cells. More High 5 cells were infected as incubation con-tinued and over 50% of the cells showed OBs in thenuclei on day 5 post-transfection. When medium frominfected High 5 cells was used to inoculate the two celllines, High 5 showed viral infection by production ofOBs in the nuclei at day 2 post-infection. The infectionlevel was above 95% at day 3 post-infection (Wgure notshown). Infection in SF-21 was diYcult to observe. Atday 5, some cells (about 3%) showed OBs in the nuclei ofinfected cells but continuing incubation resulted in celldeath, which obscured the display of OBs in infectedcells. It is likely that more cells than those showing OBsin the nuclei were infected, but this cell type did notappear to support the formation of OBs. Nevertheless,SF-21 cells are most likely permissive to ThorMNPV,but at a low level.

3.4. Biological activity of ThorMNPV

Out of seven noctuid insects tested for susceptibilityto ThorMNPV, only two, P. includens and T. ni, weresuccessfully infected (Table 1). Light microscope obser-vation of infected larvae showed large numbers of poly-hedral-shaped OBs.

These studies showed that ThorMNPV can causemortality in P. includens and T. ni at low dosages(Table 1). The LD50s of ThorMNPV to third instarP. includens and T. ni were 242 and 19 OBs per larva,respectfully, in the leaf disc assay. The LD50 of a refer-ence virus, AcMNPV, to third instar T. ni was 30 OBsper larva. The LD50 of ThorMNPV against third instarT. ni was signiWcantly lower than against P. includens atthe 95% signiWcance level, but there was no diVerence

X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135 131

between ThorMNPV and AcMNPV in infectivityagainst third instar T. ni (Table 1).

3.5. Occlusion body morphology and histopathology of ThorMNPV

Scanning electron microscopy revealed thatThorMNPV has a polyhedral, or more precisely, adodecahedral-shaped polyhedron with a size range of0.73–1.87 �m and a mean and standard error of1.3 § 0.5 �m (n D 60) (Fig. 5A). TEM studiesdemonstrated that this NPV isolated from T. orichal-cea is a multicapsid NPV (Figs. 5B–D). The numbers of

nucleocapsids per virion varied from 1 to 8, and thedominant numbers of nucleocapsids per virion were3–6.

Fat body tissue was the major site for the replicationof ThorMNPV in P. includens and T. ni. However, thevirus was also observed in tracheal epithelium cells(Figs. 5B–D). The virogenic stroma (VS) was foundassociated with the assembly of the viral nucleocapsidsand the nucleocapsid envelopes (Figs. 5B and D).Nucleocapsids could be seen at various stages of enve-lope formation (Figs. 6A–C). The VS was observedconcurrent with polyhedron formation (Fig. 5B). LargeWbrous sheets (FS) were produced in both the nuclei

Fig. 3. Nucleotide sequence of ThorMNPV polyhdrin gene and Xanking region. Above the nucleotide sequence is the deduced amino acid sequenceof the polyhedrin gene. Putative transcription start (TAAG) and polyadenylation (AATAAA) sites are shown in bold. Underlined is the polyhedringene promoter sequence which is identical to that of AcMNPV (Hooft van Iddekinge et al., 1983).

132 X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135

and cytoplasm of infected tracheal epithelium cells(Figs. 5B and C). Polyhedral envelopes were formedconcurrent with the formation of polyhedrin aroundthe virions (Fig. 5D). Most of the nucleocapsids werenormal length straight rods (Figs. 5A–C). However,abnormal forms such as curved (Figs. 6D and E) andelongated (Fig. 6F) were also observed.

Fig. 4. Neighbor joining distance tree based on concatenated aminoacid sequences of partial polh/gran, lef-8, and lef-9 genes of diVerentgroup I NPVs, including Autographa californica MNPV, AcMNPV(GenBank L22858); Junonia coenia NPV, JucoNPV (AY519235–36);Bombyx mori NPV, BmNPV (L33180 und AY519216–18); Rachiplusiaou NPV, RoMNPV (NC_004323); Coloradia pandora NPV, CopaNPV(AY519228–30); Pterolocera amplicornis NPV, PtamNPV(AY519255–57); Aporia crataegi NPV, ApcrNPV (AY519210–12);Epiphyas postvittana NPV, EppoNPV (NC_003083); Phryganidia cali-fornica NPV, PhcaNPV (AY519249–51); Orgyia pseudotsugata NPV,OpMNPV (U75930); and Choristoneura fumiferana NPV, CfMNPV(NC_004778) and ThorNPV. The sequence of the group II Spodopteraexigua NPV, SeNPV (AF169823) was used as outgroup for rooting thegroup I NPV tree. The partial polh sequences of AcMNPV andJucoNPV were omitted from the analyses due to the mosaic structureof these genes (Jehle, 2004). Numbers at the nodes indicate bootstrapvalues (each 1000 replicates) of maximum parsimony (MP), minimumevolution (ME) and neighbor joining (NJ) analyses, respectively( D MP/ME/NJ).

4. Discussion

PuriWcation of virions of baculoviruses is mostlyaccomplished by sucrose gradient centrifugation (Harrapet al., 1977; Kawanishi and Paschke, 1970; Wang andMcCarthy, 1993). The number of bands formed by anNPV depends on the maximum number of nucleocapsidsper envelope (Kawanishi and Paschke, 1970). Tanadaand Kaya (1993) reported that the number of nucleo-capsids per virion may range from 1 to 29. For ThorNPV,the number of virion bands in sucrose gradients wasabout 8 (data not shown). This number was conWrmed byultrastructure studies (data not shown). The banding pro-Wle of a baculovirus may be intrinsic to the virus itself,but, the genetic control of the number of nucleocapsidper virion envelope remains to be elucidated.

ThorMNPV was characterized in heterologous hosts,P. includens and T. ni, but was deemed to be an isolatefrom T. orichalcea. An NPV previously isolated from P.includens was a single nucleocapsid NPV (Livingstonand Yearian, 1972), whereas both single and multipleenveloped NPVs were isolated from T. ni (Fielding andDavison, 1999; Heimpel and Adams, 1966). A compari-son of the EcoRI restriction proWle of ThorMNPV withthat reported for TnMNPV by Smith and Summers(1979), showed obvious diVerences between the twoviruses. In addition, the ThorMNPV polyhedrin genesequence was distinguishable from others in GenBank.Another reason for assigning this virus to T. orichalceais that no ThorMNPV was retrieved when a second ship-ment of ThorSNPV from T. orichalcea was propagatedin P. includens and T. ni colonies, (Cheng and Carner,2000). This suggests that ThorMNPV was not a latentvirus in the colonies of either P. includens or T. ni. Theearlier sample was mostly composed of ThorSNPV andhad a minimum amount of ThorMNPV that was notdiscovered by microscopic examination. P. includensproved to be a good host since it was more susceptible toThorMNPV than to ThorSNPV (Table 1; Cheng andCarner, 2000). However, ThorSNPV was still able to

Table 1Infectivity and host range tests of ThorMNPV¤

¤ LD values in the table were calculated with a computer program POLO-PC¤¤ LD50 with same letters indicate that they are not signiWcant diVerent at 95% level. LD50 with diVerent letters indicate that they are signiWcant

diVerent at 95% level.¤¤¤ N/A (not applicable) indicates no mortality was produced.

Viruses Hosts LD50 (OBs per larva)¤¤ 95% conWdence limits Slope

Upper Lower

AcMNPV T. ni 30.90a 48.40 19.90 1.25ThorMNPV T. ni 16.95a 53.91 5.52 1.41ThorMNPV P. includens 242.46b 423.06 133.60 1.68ThorMNPV S. exigua N/A¤¤¤ N/A N/AThorMNPV S. frugiperda N/A N/A N/AThorMNPV S. eridania N/A N/A N/AThorMNPV A. gemmatalis N/A N/A N/AThorMNPV H. zea N/A N/A N/A

X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135 133

replicate in P. includens at a minimum level in the mixedinfections. This raises the possibility that both virusesmay always be present together. Tests of the two NPVson the original host, T. orichalcea, were not performeddue to the unavailability of insects. Based on the polh,lef-8, and lef-9 phylogenetic analyses and pathology, it isappropriate to name this ThorMNPV under the presentbaculovirus nomenclature scheme.

Mixed infections by viruses are common in Weld col-lected insects (Steinhaus, 1957; Vago, 1959) and it islikely that ThorSNPV and ThorMNPV can cause mixedinfections in T. orichalcea. In laboratory observations,ThorSNPV and ThorMNPV replicated in the same tis-sues in P. includens, e.g., fat body and tracheal epithelialcells. During mixed infections, both types of virusescould be observed in the same tissues, but not in thesame cells (data not shown).

The genome size (136 kb) of ThorMNPV is similar tothat of the ThorSNPV and AcMNPV (Ayres et al., 1994;Cheng and Carner, 2000). Genome size in baculovirusescan range from 81 to 160 kb (Blissard and Rohrmann,1990; Lauzon et al., 2004; Mathews, 1982), and is rela-tively stable and a reliable characteristic for a givenvirus. The REN proWle for a baculovirus is also rela-tively stable and is often used as an eVective tool todiVerentiate closely related viruses. By using thistechnique, Harrison and Bonning (1999) were able topresent evidence that the NPVs of Rachiplusia ou and

Anagrapha falcifera were variants of the same virus.REN proWles of the two NPVs in this study were quitediVerent. This characteristic, along with diVerences inmorphology and nucleotide sequence, indicate that thetwo NPVs are not closely related (Fig. 4).

ThorMNPV showed high virulence to third instar P.includens in the leaf disc bioassay with an LD50 of242 OBs per larva (Table 1). This was 270 times morevirulent than the ThorSNPV against the same host at thesame larval stage (Cheng and Carner, 2000). The LD50 ofThorMNPV against third instar T. ni is 19 OBs perlarva, which is similar to that of AcMNPV against thesame host (30 OBs per larva) (Cheng et al., 1998). How-ever ThorMNPV has a narrower host range comparedto AcMNPV. Considering the virulence of ThorMNPVagainst T. ni, this virus may have promise as a microbialinsecticide against looper species including the loopercomplex found in Indonesia and other countries ofSoutheast Asia. ThorMNPV was not found to causemortality in other caterpillars tested. For example, S.frugiperda was a resistant insect. However, ThorMNPVmay be able to replicate in larvae of S. frugiperda sincethere was evidence of ThorMNPV infection in SF-21cells which originated from S. frugiperda.

Fibrous sheets (FS) were observed in the nucleus andthe cytoplasm of epithelial cells in the trachea of P. inclu-dens infected with ThorMNPV (Figs. 5B–D). The FS arebelieved to be of protein origin and coded by a viral gene

Fig. 5. Electron microscopic investigation of ThorMNPV. Scanning electron micrograph (15 kV, 7000£) showing the dodecahedral shape of Thor-MNPV polyhedra (A). Transmission electron micrograph of the tracheal epithelium cells of P. includens : formation of Wbrous sheet (FS) by Thor-MNPV in the nucleus (B) and in the cytoplasm (C). Formation of polyhedron envelope (PE) of ThorMNPV in the nucleus of P. includens trachealepithelium cells (D). VS, virogenic stroma; NE, nuclear envelope; and T, taniedium. Bar D 1 �m.

134 X.-W. Cheng et al. / Journal of Invertebrate Pathology 88 (2005) 126–135

called p10 (Smith et al., 1982, 1983). This gene may func-tion in the release of polyhedra from infected cells at theend of infection (Van Oers et al., 1993) and in the sealingof polyhedral envelopes (Gross et al., 1994). Van Oersand Vlak (1997) stated that the p10 gene may give bac-uloviruses an evolutionary advantage by enabling themto spread viral progeny and therefore continue the infec-tion to other host insects.

In conclusion, based on pathology, REN analysis andpolh, lef-8, and lef-9 sequence comparisons, ThorMNPVis a new isolate of Group I NPVs of Baculoviridae. Thehigh virulence of ThorMNPV to T. ni and P. includenssuggests its potential as a viral insecticide. The availabil-ity of a permissive cell line (High 5) for DNA transfec-tion and viral infection should allow plaque puriWcationof ThorMNPV and genetic engineering of the virus toenhance its eVectiveness.

Acknowledgments

The authors thank Dr. G. F. Rohrmann for providingthe OpMNPV polyhedrin gene clone. Financial support

was provided by HATCH Project 1614, by a USAIDProject, Integrated Pest Management Research, Devel-opment and Training Activities for Palawija Crops inIndonesia and the College of Arts and Science new fac-ulty startup fund to XWC at Miami University.

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