Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1-1-2002

Polyhedral envelope protein mutants of ou multi- nucleocapsid nucleopolyhedrovirus

Hailing Jin Iowa State University

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Recommended Citation Jin, Hailing, "Polyhedral envelope protein mutants of multi-nucleocapsid nucleopolyhedrovirus" (2002). Retrospective Theses and Dissertations. 20111. https://lib.dr.iastate.edu/rtd/20111

This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Polyhedral envelope protein mutants of Rachiplusia ou multi-nucleocapsid

nucleopolyhedrovirus

by

Hailing Jin

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Genetics

Program of Study Committee: Bryony C. Bonning, Major Professor Susan L. Carpenter Jeffrey K. Beetham

Iowa State University

Ames, Iowa

2002 11

Graduate College Iowa State University

This is to certify that the master's thesis of

Hailing Jin has met the thesis requirements of Iowa State University

Signatures have been redacted for privacy 111

TABLE OF CONTENTS

LIST OF FIGURES lV

LIST OF TABLES v

ABSTRACT Vl

CHAPTERl.GENERALINTRODUCTION 1 LITERATURE REVIEW 1 Baculovirus biology 1 Baculoviruses as biological control agents 5 Recombinant baculoviruses in pest management 7 Strategies for reduction of lethal dose 10 THESIS ORGANIZATION 13

CHAPTER2. POLYHEDRAL ENVELOPE PROTEIN MUTANTS OF RACH/PLUS/A OUMULTI-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS 14 ABSTRACT 14 INTRODUCTION 15 MATERIALS AND METHODS 16 RESULTS 24 DISCUSSION 28 ACKNOWLEDGEMENTS 33

CHAPTER 3. GENERAL DISCUSSION 65

REFERENCES 68

ACKNOWLEDGEMENTS 84 lV

LIST OF FIGURES

FIG.1. Construction ofRoPEPLacZ and RoPEP~ATG-TAG. 35

FIG.2. Alignment ofbaculovirus polyhedral envelope protein sequences. 37

FIG.3. Unrooted phylogram derived from analysis of baculovirus PEP proteins. 40

FIG.4. Analysis of recombinant virus genomes by restriction enzyme digestion. 42

FIG.SA. Analysis of virus proteins in cultured cells by SDS-PAGE. 44 SB. Western blot analysis of PEP expression in cultured cells.

FIG.6. Scanning electron micrographs of polyhedra. 47

FIG.7. Transmission electron micrographs of polyhedra. 49

FIG.8. Immunogold labeling of PEP. 51

FIG.9. Sensitivity of polyhedra to alkali. 54

FIG.10. Ostrinia nubilalis and Trichop/usia ni midgut pH analysis. 57

FIG.11. Comparison of the number ofvirions encapsulated within polyhedra. 60

FIG.12. Preliminary examination of the numbers ofnucleocapsids in occlusion- 62 derived virions for pep-mutant and control viruses. v

LIST OF TABLES

TABLE 1. Baculovirus genomes sequenced. 5

TABLE 2. Lethal concentration data for and pep-mutant RoMNPV in first instar 63 Ostrinia nubilalis and Trichoplusia ni.

TABLE 3. Ratios of band intensities for ODV containing different numbers of 64 nucleocapsids. Vl

ABSTRACT

Baculoviruses are -specific, double-stranded DNA viruses, and have great potential as environmentally friendly biopesticides. Rachiplusia ou multi-nucleocapsid nucleopolyhedrovirus (RoMNPV), also known as Anagraphafa/cifera multi-nucleocapsid nucleopolyhedrovirus (AfMNPV), can infect more than 31 species of , and has an attractive host range for insect pest control purposes. We investigated whether inactivation of the baculovirus pep gene would increase the virulence ofRoMNPV against lepidopteran larvae. The RoMNPV pep gene encodes the polyhedral envelope protein (PEP), which surrounds polyhedra. PEP is the main protein component of the polyhedral envelope. The pep gene is unnecessary for viral replication. The RoMNPV pep gene was inactivated by insertion of the /acZ gene to produce the recombinant virus RoPEPLacZ. Site-directed mutagenesis was used to produce two viruses with single mutations, RoPEPAATG and RoPEPTAG, and one virus with both mutations, RoPEPMTG-TAG. These mutations were designed to inactivate pep. Western blot analysis indicated that normal pep gene expression was disrupted for all pep mutant viruses except RoPEPAATG, which expressed wild-type levels of PEP. Scanning, transmission, and immunogold electron microscopy indicated that the polyhedral envelope was absent from polyhedra ofRoPEPTAG, RoPEPAATG-TAG and RoPEPLacZ. Although immunogold electron microscopy suggested that a polyhedral envelope was present on RoPEPMTG polyhedra, scanning electron microscopy showed that polyhedra of this mutant had a pitted surface consistent with the absence of an envelope. In bioassays, pep-mutant viruses showed reduced potency against neonate larvae of a semi- permissive host, the European com borer Ostrinia nubilalis, and unaltered potency against neonate larvae of the permissive host, the cabbage looper Trichop/usia ni. Several hypotheses, including the potential effects of midgut pH, a reduction in the number of nucleocapsids per occlusion-derived virion, and a reduction in the number ofvirions per polyhedron, were tested to explain the reduction in virulence against 0. nubila/is. Data showed that there were significantly fewer virions per polyhedron for all pep-mutant viruses compared to the control, which may contribute to the reduced virulence of the pep-mutant viruses against semi-permissive species. 1

CHAPTERl. GENERAL INTRODUCTION

LITERATURE REVIEW

Baculovirus biology

Baculoviruses are double-stranded DNA viruses and have a large covalently closed circular DNA genome of90-160 kb. Baculoviruses belong to the family Baculoviridae that is divided into two genera: nucleopolyhedroviruses (NPV) and granuloviruses (GV) (Volkman et al., 1995). The NPV produce large polyhedron-shaped occlusion bodies called polyhedra that contain many virions (occlusion-derived virions ), while the GV have smaller occlusion bodies called granules that normally contain only one virion per granule (Adams & Bonami, 1991).

Baculovirus lifecycle Baculoviruses have a unique bi-phasic lifecycle, which is temporally regulated. Two morphologically and biochemically distinct infectious viral phenotypes are produced during the lifecycle, occlusion-derived virus (ODY) and budded virus (BV). ODY is responsible for horizontal transmission between insect hosts (Evans, 1986), while BV is responsible for systemic spread through the insect host and propagation in tissue culture (Entwistle & Evans, 1985; Keddie et al., 1989). Infection occurs following ingestion of occlusion bodies by insect larvae. The occlusion bodies are dissolved in the alkaline environment of the midgut of Lepidoptera (pH9.5-11.5), and virion particles are released into the gut lumen (Entwistle & Evans, 1985; Granados, 1980). The fore- and hindgut of are derived from embryonic endodermal cells and are lined with cuticle. Only the midgut derived from the endoderm and not lined with cuticle is involved in primary virus attachment and entry. Occlusion derived virions cross the peritrophic matrix, an extracellular layer of protein and chitin that separates food in the midgut lumen from the midgut epithelium (Lehane, 1997), and initiate infection by binding to the columnar epithelial cells and entering the tips of the microvilli on the apical 2

brush border of cells. Following fusion with the cell membrane (Horton & Burand, 1993), the nucleocapsids are released into the cytoplasm and are transported to the nuclei where viral DNA replication and production of nucleocapsids ensues. Progeny nucleocapsids pass into the cytoplasm, are transported to the basal membrane of the midgut cells and bud through the basal membrane. Budded virus is disseminated to other tissues within the insect by passage through the tracheal system of the insect (Engelhard et al., 1994). The NPV infect virtually all tissues of the host insect, while the tissue tropism of the GV tends to be more limited (Federici, 1997). Occlusion bodies are produced in all infected cells. Larvae become pale in color, cease feeding and show limited movement. Finally, infected larvae die and release occlusion bodies into the environment following liquefaction of the cadaver and rupture of the insect cuticle.

Regulation of baculovirus gene expression Baculovirus gene expression is regulated in a cascade, which is divided into four phases: immediate early (starting at 0-2 hours post-infection), early (starting at 6 hours post- infection), late (starting at 10-12 hours post-infection), and very late (starting at 15 hours post-infection) (Blissard & Rohrmann, 1990). Baculovirus gene expression is regulated primarily at the level of transcription. Immediate early and early genes are transcribed with the host RNA polymerase II (Hub & Weaver, 1990). Some immediate early gene products, such as IEl, IE2, and PE38, work as trans-activators to regulate early gene expression and DNA replication (Friesen, 1997). Late and very late genes are transcribed by a virally encoded RNA polymerase (Guarino et al., 1998). Nucleocapsid structural proteins, such as vp39 and p6.9, are expressed during the late phase of the infection cycle. Polyhedral structural proteins are expressed during the late and very late phases. There are at least three proteins involved in forming the polyhedra: polyhedrin, p 10 and the polyhedral envelope protein (Russell et al., 1991).

Structural proteins ofbaculovirus polyhedra All nucleopolyhedroviruses (NPV) examined have polyhedral envelope proteins (PEP). This protein is found around the periphery of the polyhedron and forms an 3

amorphous, electron-dense layer (or calyx), which was thought to be composed of carbohydrate (Minion et al., 1979). PEP was first identified from Autographa californica multi-nucleocapsid nucleopolyhedrovirus (AcMNPV) (Whitt & Manning, 1988). AcMNPV PEP has 322 amino acid residues, is phosphorylated, and has a molecular weight of 34kDa (Lent et al., 1990). The Orygia pseudotsugata MNPV (OpMNPV) pep gene is located in the HindlII-M fragment and encodes a 297 amino acid polypeptide (Gombart et al., 1989). OpMNPV PEP shares 58% amino acid identity with AcMNPV PEP (Oellig et al., 1987). PEP appears to be covalently linked to carbohydrate via thiol bonds. PEP is not required for virus replication and may function to prevent loss ofvirions from polyhedra and to prevent aggregation of polyhedra (Gross, 1994; Oers & Vlak, 1997; Whitt & Manning, 1988). OpMNPV PEP was first detected 24 hours post-infection by western blot, and expression increased from 36 to 72 hours post-infection (Gombart et al., 1989). PEP was not associated with the periphery of developing polyhedra until 72 hours post-infection, and appears as a part of the mature polyhedral envelope at 96 hours post-infection (Russell & Rohrmann, 1990). The reporter enzyme, {J-glucuronidase (GUS), was expressed under control of the pep or pl 0 promoter (Gross, 1994). Analysis of GUS activity showed that the pep promoter is one of the weakest baculovirus late gene promoters. Homologs of PEP are not found in granuloviruses (GVs) even though a similar electron-dense envelope structure has been observed (Longworth et al., 1972). Polyhedrin is the major constituent of the polyhedral matrix and has a molecular weight of 29 kDa (Rohrmann, 1986; Rohrmann, 1992). Based on genomic data, polyhedrin is the most highly conserved of the baculovirus proteins. AcMNPV polyhedrin shares 89% amino acid identity with Orgyia pseudotsugata multi-nucleocapsid nucleopolyhedrovirus (OpMNPV) (Ahrens et al., 1997). Alignment of the polyhedrin genes from 20 different NPVs isolated from Lepidoptera, showed that the amino acid identity is typically around 70%, with the highest identities of 97-99% (Chou et al., 1996). NPVs are subdivided into two groups, group I and group II, based on molecular phylogenetic analysis ofpolyhedrin sequences (Zanotto et al., 1993). The two groups have distinct evolutionary rates. Group I NPVs are closely related, while group II NPV s are more divergent. 4

The p 10 protein is linked to fibrillar structures that are abundant in the nucleus and cytoplasm of baculovirus infected cells. It has a molecular weight of 10 kDa. The function of plO is unclear (Oers & Vlak, 1997). The plO protein is poorly conserved in NPVs (Zuidema et al., 1993). Alignment of plO sequences from six baculoviruses showed the presence of amphipathic a-helix structures at the N-terminus (Wilson et al., 1995). These helices are involved in formation of coiled-coil structures that are associated with protein oligomerization domains.

Baculovirus genome analysis Autographa californica multi-nucleocapsid nucleopolyhedrovirus (AcMNPV) has been widely studied, and most data on baculovirus structure, pathogenesis, morphogenesis and genomics come from studies on AcMNPV. There are several clones of AcMNPV, including clone C6 (Possee et al., 1991), clone E2 (Smith & Summers, 1979), clone E (Tjia et al., 1979), clone HR3 (Cochran et al., 1982), and clone Ll (Miller & Dawes, 1979). The whole nucleotide sequence of clone C6 has been determined and analyzed (Ayres et al., 1994). The DNA genome has 133,894 base pairs, and the overall A+T content is 59%. The genome was predicted to contain 154 methionine-initiated open reading frames (ORFs) encoding peptides of more than 50 amino acids. These ORFs are evenly distributed on the two strands of DNA, and 59 of the 154 predicted ORFs share homology with other genes. Rachiplusia ou multi-nucleocapsid nucleopolyhedrovirus (RoMNPV) was first isolated from the mint looper, Rachiplusia ou in 1960 (Paschke & Hamm, 1961; Paschke & Sweet, 1966). Sequencing of the EcoRI-G fragment (which contains the polyhedrin gene) of RoMNPV and comparison with the corresponding region of AcMNPV indicated that RoMNPV is closely related to AcMNPV (Harrison & Bonning, 1999). Restriction digestion and nucleotide sequence data also revealed that RoMNPV is identical to the Anagrapha falcifera multi-nucleocapsid nucleopolyhedrovirus (AfMNPV) (Chen et al., 1996). The whole genome ofRoMNPV has been sequenced (Dr. Robert Harrison, Iowa State University, unpublished). The complete genomes of several other baculoviruses have been sequenced and analyzed in recent years: 5

Table 1. Baculovirus genomes sequenced. Virus Reference Epiphyas postvittana MNPV (EppoMNPV) (Hyink et al., 1998) Orgyia pseudotsugata MNPV (OpMNPV) (Ahrens et al., 1997) Bombyx mori MNPV (BmMNPV) (Gomi et al., 1999) Helicoverpa armigera SNPV (HaSNPV) (Chen et al., 2001) Lymantria dispar MNPV (LdMNPV) (Kuzio et al., 1999) Spodoptera exigua MNPV (SeMNPV) (ljkel et al., 1999) Spodoptera litura MNPV (SpltMNPV) (Pang et al., 2001) Cu/ex nigripalpus NPV (CuniNPV) (Afonso et al., 2001) Xestia c-nigram GV (XcGV) (Hayakawa et al., 1999) Piute/la xylostella GV (PxGV) (Hashimoto et al., 2000) Cydia pomonella GV (CpGV) (Luque et al., 2001)

Baculoviruses as biological control agents

Baculoviruses are arthropod-specific, and harmless to vertebrates, plants and other non-target organisms. Baculoviruses primarily infect the Lepidoptera ( and butterflies), but have been identified in Hymenoptera, Diptera, Coleoptera, Neuroptera, and Trichoptera, as well as in Crustacea (Anderson & Prior, 1992; Chang et al., 1993; Couch, 1991; Granados & Federici, 1986a; Granados & Federici, 1986b). Baculoviruses have been isolated from more than 500 species of the Lepidoptera, with most being infectious to only a single , or to a few species within a family. RoMNPV and AcMNPV have broader host ranges and are known to infect more than 30 species within the Lepidoptera. Baculoviruses have a long history of use as biological control agents. The nun , Lymantria monacha, was controlled by a nucleopolyhedrovirus in pine forests in Germany in 1892 (Huber, 1986). Diprion hercyniae nucleopolyhedrovirus (HtgNPV) was used to control the European spruce sawfly in North America in 1930 (Bird & Burk, 1961). One of the most successful examples is the use of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) to control the soybean looper, Anticarsia gemmatalis, in Brazil. AgMNPV has been used 6

effectively in soybean fields since 1981 and approximately one million hectares is treated with AgNPV annually (Moscardi, 1999). Baculoviruses are currently successfully applied to protect forests and a variety of crops worldwide (Inceoglu et al., 2001 ).

Limitations of using baculoviruses as insecticides Although baculoviruses have shown great potential as environmentally benign alternatives to synthetic chemical pesticides, their use is still very limited (Moscardi, 1999). The main factors that limit the use of baculoviruses are: (1) low stability in the field, (2) slow killing speed, (3) high production costs, and ( 4) narrow host specificity. ODVs (the phenotype that is infectious to larvae) are embedded in polyhedra and are stable for years in the soil (Jaques, 1985). The persistence and viability of virus are greatly affected by environmental factors, and particularly by UV radiation. UV-B (280-320nm) and UV-A (320-400nm) induce mutation of viral DNA thereby blocking normal DNA synthesis, resulting in virus deactivation. Baculoviruses have an average half-life of 2-5 days, and are completely deactivated by 24 hours of exposure to UV-light (Jaques, 1985). Temperature has less of an effect on baculovirus viability than UV radiation. Low (around 20°C) or high (around 39°C) temperatures inhibit virus replication. Peak mortality caused by AgNPV is delayed by 4 days at 20°C compared with 27°C (Moscardi, 1999). The slow killing speed is a major disadvantage for commercialization ofbaculovirus insecticides. The time taken from ingestion of polyhedra to infection, death of the host insect, and release of progeny virus is highly variable for different host-virus combinations. Many factors affect the speed of virus-induced mortality, including the larval instar infected, the condition of the larvae, the virus dose received, the virulence of the virus, environmental temperature and the diet of the host insects. Usually it takes 2-5 days for baculoviruses to kill permissive hosts, and 2-3 weeks to kill semi-permissive hosts (Ignoffo, 1966). During this period, virus-infected insects continue to consume crops causing considerable economical loss. Such losses make baculoviruses weak competitors for the fast-acting chemical pesticides. There are large-scale methods for both in vivo and in vitro production of baculoviruses. Both methods are costly resulting in increased prices ofbaculovirus products. 7

Mass rearing of insects on artificial diets and virus infection of insect larvae are labor- intensive procedures, although automated systems have been developed (Hawtin & Possee, 1993). In Brazil, AgNPV-infected larvae are collected form the field since labor there is relatively cheap (Moscardi & Sosa-Gomez, 1992; Moscardi & Sosa-Gomez, 1996). It is a challenge to control product quality and to avoid contamination of in vivo cultures. Batch culture in large sterile airlift fermenters has been developed and improved for recombinant baculovirus production in vitro, but economical cell culture techniques have not been developed for agricultural use due to the high cost of insect cell culture medium (Sheih, 1989). The use of serum-free medium could greatly reduce production costs since serum is the most expensive component of the cell culture medium. Host specificity is desirable for use of insect management products in integrated pest management (IPM) because the impact is less than that of chemical pesticides on natural enemies and non-target organisms (Fuxa, 1990). However, the large agricultural companies are not interested in products for small markets. In addition, growers prefer not to have to use several control agents to control different pest insects in the same cropping system.

Recombinant baculoviruses in pest management

With the incredible progress in biotechnology over the past two decades, our increased knowledge ofbaculovirus gene structure, function and regulation has allowed researchers to overcome some of the unfavorable properties ofbaculoviruses as biological control agents. Most of the research has focused on increasing the killing speed of baculoviruses, thereby reducing fee~ing damage caused by virus-infected larvae. Strategies have included genetic engineering of virus genomes to express genes encoding insect hormones, enzymes, and insect-specific toxins, and deletion of specific viral genes to enhance pesticide efficacy (Harrison & Bonning, 2000; Van Beek & Hughes, 1998).

Recombinant baculoviruses expressing insect hormones Insect hormones play an important role in regulation of insect development and physiological activity. Disruption, untimely- or over-expression of insect hormones results in 8

abnormal growth, changes in feeding behavior, and even death. Insect hormones have been produced and applied directly as insecticides in the field. One strategy used to enhance the properties ofbaculovirus insecticides has been to express insect hormones to disrupt the normal hormonal balance of the infected insects. For example, diuretic and anti-diuretic hormones function in regulation of water balance. The diuretic hormone (DH) coding sequence of the tobacco homworm, Manduca sexta, was cloned into BmNPV to produce the recombinant baculovirus BmDH5. Bioassay results showed that BmDH5 killed larvae 20% faster then BmNPV. BmDH5 also caused a 30% decrease in hemolymph volume compared with the control (Maeda, 1989). The eclosion hormone (EH) of M sexta was expressed in a baculovirus without any significant effect on the efficacy of the virus (Eldridge et al., 1992); prothoracicotropic hormone (PTTH) of B.

mori was expressed and resulted in an inexplicable increase in the LC50 compared to the virus (O'Reilly et al., 1995).

Deletion of endogenous baculovirus genes Deletion of a baculovirus gene is another approach to enhancing the insecticidal properties ofbaculoviruses. The AcMNPV egt gene encodes an enzyme, ecdysteroid glucosyl transferase (ETG). EGT is a 506 amino acid polypeptide with a molecular weight of 57kDa that initiates the transfer of glucose (or galactose) from UDP-glucose to ecdysteroids. This transfer blocks molting of the insect host by inactivating ecdysteroids that trigger ecdysis (O'Reilly & Miller, 1989). AcMNPV infected larvae have a prolonged larval stage, which results in increased yield of progeny virus (O'Reilly & Miller, 1991)). The egt gene may have been acquired by the virus from an insect host (O'Reilly & Miller, 1990). Deletion of the egt gene from the AcMNPV genome resulted in a 30% improvement in killing speed and a 40% reduction in food consumption compared to larvae infected with AcMNPV. In addition, larvae infected with egt-negative viruses do not leave the host plant to pupate, thereby enhancing the opportunity for virus spread to other larvae. The mechanism by which egt deletion increases killing speed is unclear. EGT may induce a hormonal effect that increases virus-specific protein synthesis and results in faster virus transmission (Kang et al., 2000). 9

Recombinant baculoviruses expressing insect-specific toxins A number of insect-specific neurotoxins have been expressed in baculoviruses. Recombinant baculoviruses expressing insect-specific neurotoxins offer some of the most promising prospects for baculovirus commercialization. The neurotoxin AaIT from the North African scorpion, Androctonus australis, was expressed in AcMNPV (McCutchen et al., 1991; Stewart et al., 1991). AaIT alters the neuronal sodium channel conductance in insects leading to pre-synaptic excitation. Insects affected by AaIT show symptoms including cessation of feeding, loss of neuromuscular control, paralysis, falling from the plant, and eventually death (Hoover et al., 1995). Recombinant AcMNPV expressing AaIT reduced feeding damage by 50% and reduced lethal time by 25% relative to virus-infected larvae (Stewart et al., 1991). Small scale field trials in the UK showed the feeding damage was reduced by 25% on cabbage plants treated with recombinant viruses compared with AcMNPV (Cory et al., 1994). Large-scale field trials were performed by American Cyanamid Company in the United Stated with two recombinant baculoviruses, Ac-AaIT and Hz-AaIT. Both Ac-AaIT and Hz-AaIT were constructed by deletion of the ecdysteroid glucosyl transferase (egt) gene and insertion of a 0.8 kb fragment containing the AaIT encoding region (Treacy et al., 2000). Bioassays showed that Ac-AaIT and Hz-AaIT had the same virulence (LC50) as wild-type viruses against the tobacco budworm, Heliothis virescens. The lethal time (LT5o) of Ac-AaIT and Hz-AaIT was significantly reduced compared to the wild-type viruses. Three field trials demonstrated that Hz-AaIT gave control of the heliothine complex in cotton equivalent to that of commercial Bt and other insecticide products. Numerous other insect-specific toxins have been cloned and expressed in baculoviruses, such as Lqh!T from the Israeli yellow scorpion Leiurus quinquestriatus hebra~us (Chejanovsky et al., 1995; Gershburg et al., 1998; Herrmann et al., 1995), µ-Aga- IV from the web spider Agelenopsis aperta (Prikhod'ko et al., 1998; Prikhod'ko et al., 1996), tox34 from the straw itch mite, Pyemotes tritici (Tomalski & Miller, 1991; Tomalski & Miller, 1992), TalTX-1 from the spider Tegenaria agrestis, and DTX9.2 from the weaving 10

spider, Diguetia canities (Hughes et al., 1997). These toxins could further increase the efficacy ofbaculoviruses for pest control.

Recombinant baculoviruses expressing insect derived enzymes Juvenile hormone (JH) is involved with regulation of insect larval development, metamorphosis, reproduction and onset of metamorphosis at the final larval molt (Riddiford, 1994). Juvenile hormone esterase (JHE) that degrades JH has potential for use in insect pest control. The cDNA sequence of JHE was cloned from the tobacco budworm, Heliothis virescens, and expressed in a recombinant baculovirus (Bonning & Hammock, 1992; Hammock et al., 1990). No significant effect on insecticidal efficacy of the recombinant virus was observed compared to the wild-type virus. Modification by mutation of specific residues of JHE dramatically increased the insecticidal efficacy in Trichoplusia ni and H virescens (Bonning et al., 1995; Bonning et al., 1997; Bonning et al., 1999). Basement membrane-degrading enzymes such as cathepsin L from Sarcophaga peregrina have also been expressed to enhance the speed of kill of recombinant baculoviruses (Harrison & Bonning, 2001). These recombinant baculoviruses are among the most effective recombinant baculoviruses for reduction in feeding damage caused by infected larvae. Other enzymes not only from insects but also from plants have been used to enhance the insecticidal performance of baculoviruses. For example, chitinase from Manduca sexta expressed by a recombinant baculovirus resulted in a reduction of the ST50 by 22-23% against Spodopterafrugiperda (Gopalakrishnan et al., 1995); expression ofURF13 from maize resulted in a reduction of ST so of 40% against T. ni (Korth & Levings, 1993).

Strategies for reduction oflethal dose

Relatively little attention has been paid to reduction of the lethal dose of baculoviruses by genetic means compared to reduction of the lethal time of the virus and reduction of feeding damage caused by infected larvae. Reduction of the virus dose required to kill the host insect would not only reduce production costs (because less virus would need 11

to be applied), but would also expand the range of pest species that could be targeted with a single virus. Enhancement of virulence would increase the likelihood that pest species that are semi-permissive to infection could be controlled. In addition to genetic optimization of baculoviruses for enhanced virulence, several enzymes and chemicals have been shown to enhance virulence when included in the baculovirus formulation.

Optical brighteners The optical brighteners are stilbene disulfonic acid derivatives that can absorb radiation in the nonvisible UV part of the electromagnetic spectrum and emit light in the blue portion of the visible spectrum. Optical brighteners were tested as possible UV protectants for baculoviruses, but unexpectedly showed the ability to enhance baculovirus-induced mortality (Shapiro & Robertson, 1992). The most well studied optical brightener for baculovirus enhancement is M2R {Tinopal LPW, Fluorescent Brightener 28, Calcofluor white M2R). M2R significantly enhanced the virulence of slower acting baculoviruses, such as Spodoptera frugiperda MNPV (S:tMNPV) (Hamm & Shapiro, 1992), Agrotis ipsilon MNPV (Boughton et al., 2001), and LdMNPV (Shapiro, 1992). The presence of 1% (w/v) M2R reduced the LC50 of LdMNPV 214-fold compared to LdMNPV alone (Dougherty et al., 1995; Dougherty et al., 1996). There are two proposed mechanisms of action for the baculovirus virulence-enhancing properties of optical brighteners. First optical brighteners change the pH of the insect midgut and block sloughing of infected midgut cells (Sheppard et al., 1994; Washburn et al., 1998). This mechanism would increase the probability of primary infection in the insect midgut. Second, several optical brighteners have been shown to bind to chitin thereby interfering with chitin fibrillogenesis. This interference results in disintegration of the peritrophic matrix, which acts as a physical barrier against pathogens (Wang & Granados, 2000).

Expression of enzymes for degradation of the peritrophic matrix The peritrophic matrix is an extracellular structure consisting of protein, chitin and glycosaminoglcans that seperates food in the midgut lumen from the midgut epithelial cells (Lehane, 1997). The peritrophic matrix has putative roles in facilitation of the digestive 12

process and as a physical barrier to prevent infection of the host by microbial pathogens (Tellam, 1996). Enhancin, a zinc metalloprotease isolated from Trichoplusia ni granulovirus (TnGY), was reported to degrade a mucin-like protein in the peritrophic matrix. Enhancin facilitates passage ofbaculoviruses through the peritrophic matrix (Lepore et al., 1996; Wang & Granados, 1997). Enhancin was shown to augment AcMNPY virulence (Wang & Granados, 1997; Wang & Granados, 1998). Application of Streptomyces griseus chitinase to the peritrophic matrix results in roughening of the surface and production of numerous holes (Brandt et al., 1978). Inclusion of an unspecified bacterial chitinase in a formulation of

LdMNPY resulted in a 5.4-fold reduction in the LC5o for host larvae (Shapiro et al., 1987). An AcMNPY chitinase that is active with a broad pH range (3.0-10.0) has been identified (Hawtin et al., 1995; Hawtin et al., 1997; Thomas et al., 1998). Recombinant baculoviruses that express these peritrophic matrix-degrading enzymes on the surface of ODY may have enhanced virulence.

Deletion of the polyhedral envelope protein The polyhedral envelope protein (PEP) is a structural protein associated with the polyhedra. In addition to AcMNPY, PEP has been identified in RoMNPY, OpMNPY, BmMNPY, EppoMNPY, HaSNPY, LdMNPY, SeMNPY and SpltMNPY. Disruption ofpep gene expression in AcMNPY by insertion of the {J-galactosidase (lacZ) gene in phase resulted in increased sensitivity of polyhedra to alkali which was presumed to result in more ready loss of virions from polyhedra (Zuidema et al., 1989). The AcMNPY pep-mutant showed a six-fold increase in virulence (reduced LC50) against first instars of the cabbage looper, Trichoplusia ni, compared to AcMNPY (lgnoffo et al., 1995). Polyhedra of the AcMNPY pep-mutant lacked the electron-dense polyhedral envelope, and dissolved more quickly in weak alkali (0.05M Na2C03) than wild-type virus. The reduction in lethal dose may have been caused by the more rapid degradation of polyhedra facilitating release of ODY for primary infection of the host, resulting in the midgut being exposed to a larger amount of infective virus over a shorter period of time (lgnoffo et al., 1995; Zuidema et al., 1989). Normally, on dissolution of polyhedra, some virions remain trapped within the 13

polyhedral envelope. In contrast, disruption of pep gene expression in LdMNPV by partial deletion or insertion of the coding sequence for green fluorescent protein (GFP) did not result in any significant difference in virulence against the gypsy moth, Lymantria dispar (Bischoff & Slavicek, 1999).

Here, we describe production ofpep-mutants ofRoMNPV and the effects of these pep-mutants on pest species that are either permissive or semi-permissive to baculovirus infection.

THESIS ORGANIZATION

Chapter 1 of the thesis provides a general introduction to the background for my research on the baculovirus polyhedral envelope protein. Chapter 2 is entitled "Polyhedral Envelope Protein Mutants of Rachiplusia ou Multi-nucleocapsid Nucleopolyhedrovirus". This chapter consists of a manuscript that will be submitted with minor modification to the Journal of Invertebrate Pathology. Chapter 2 is co-authored by Robert L. Harrison, Diane Schroeder, Anthony J. Boughton and Bryony C. Bonning. Dr. Robert L. Harrison's contribution to the project was construction of pRoPEPD.ATG-TAG, and provision of many good suggestions. Diane Schroeder's contribution was the sequencing of the pep gene and construction ofpRoPEPLacZ. Dr. Anthony J. Boughton assisted with setup and analysis of bioassays. Dr. Bryony C. Bonning, my major professor, provided funding to make the research possible and gave extensive guidance on the project, on interpretation of results and on preparation of the manuscript. Chapter 3 contains a general discussion. All references cited are listed at the end of the thesis. 14

CHAPTER 2. POLYHEDAL ENVELOPE PROTEIN MUTANTS OF RACHIPLUSIA OUMULTI-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS

A paper to be submitted to the Journal of Invertebrate Pathology

Hailing Jin, Robert L. Harrison, Diane Schroeder, Anthony J. Boughton and Bryony C. Bonning

ABSTRACT

To increase the virulence of the baculovirus Rachiplusia ou multi-nucleocapsid nucleopolyhedrovirus (RoMNPV) against lepidopteran pests, we mutated the RoMNPV polyhedral envelope protein gene (pep) to prevent formation of the polyhedral envelope. The pep ORF was disrupted by insertion of lacZ to produce the recombinant virus RoPEPLacZ. Two additional pep-mutant recombinant viruses were produced by site-directed mutagenesis that eliminated the pep initiation codon (Ro PEP MTG) or inserted a stop codon at position

13 of the pep ORF (RoPEPTAG). A fourth pep-mutant virus, RoPEP~ATG-TAG, carried both the ablation of the pep initiation codon and the insertion of the stop codon. Western blot analysis revealed that PEP was still expressed during infection of Sf9 cells with RoPEPMTG. A greatly reduced quantity of PEP was evident in Sf9 cells infected with RoPEPTAG or Ro PEP MTG-TAG, indicating that translation occurred past the inserted stop codon. No PEP was detected in Sf9 cells infected with RoPEPLacZ. Scanning, transmission, and immunogold electron microscopy indicated that the polyhedral envelope was absent from polyhedra ofRoPEPTAG, RoPEP~ATG-TAG and RoPEPLacZ. Although immunogold electron microscopy suggested that a polyhedral envelope was still present on RoPEP~ATG polyhedra, scanning electron microscopy showed that polyhedra of this mutant had a pitted surface consistent with the absence of an envelope. In bioassays, pep-mutant viruses showed reduced potency against neonate larvae of a semi-permissive host, the European com borer Ostrinia nubilalis. The virulence of the mutant viruses was unaltered relative to RoMNPV against a permissive host, the cabbage looper Trichoplusia ni. The numbers ofnucleocapsids per virion and virions per polyhedron, were examined for mutant and viruses by sucrose 15

gradient centrifugation and EM respectively. There were no obvious differences in the numbers of nucleocapsids per virion between viruses. However, there were significantly fewer virions (7-8 per µm2 of polyhedral cross-sectional area) for all pep-mutant viruses compared to the wild-type control (12 per µm2 of polyhedral cross-sectional area). Fewer virions per polyhedron from pep-negative viruses could contribute to the reduction of virulence against semi-permissive species.

INTRODUCTION

Baculoviruses are double-stranded DNA viruses with a large covalently closed circular DNA genome of 90-160 kb. They belong to the family Baculoviridae that is divided into two genera: nucleopolyhedroviruses (NPV) and granuloviruses (GV) (Volkman et al., 1995). Baculoviruses are arthropod-specific, and harmless to vertebrates, plants and other non-target organisms. They have received considerable attention to be used as environmentally benign biopesticides. Baculoviruses have a unique bi-phasic lifecycle. Two morphologically and biochemically distinct infectious viral phenotypes, occlusion-derived virus (ODV) and budded virus (BV), are produced during the lifecycle. ODV is responsible for horizontal transmission between insect hosts (Evans, 1986), while BV is responsible for systemic spread through the insect host and propagation in tissue culture (Entwistle & Evans, 1985; Keddie et al., 1989). Rachiplusia ou multi-nucleocapsid nucleopolyhedrovirus (RoMNPV), also known as Anagrapha falcifera multi-nucleocapsid nucleopolyhedrovirus (A£MNPV) (Harrison & Bonning, 1999), was first isolated from the mint looper, Rachiplusia ou, in Indiana in 1960 (Pashke & Hamm, 1961). RoMNPV can infect more than 31 species ofLepidoptera (Hostetter & Puttler, 1991), and has an attractive host range for insect pest control purposes. The RoMNPV pep gene, which is unnecessary for viral replication, encodes the polyhedral envelope protein (PEP). This protein is distributed on the periphery of polyhedra and helps to form the polyhedral envelope structure (Gombart et al., 1989; Whitt & Manning, 1988). Disruption ofpep gene expression in AcMNPV resulted in increased sensitivity of polyhedra 16

to alkali and apparent more ready loss ofvirions from polyhedra (Zuidema et al., 1989). The AcMNPV pep-mutant showed a six-fold increase in virulence (reduced LC50) against first instar cabbage looper, Trichoplusia ni, compared to AcMNPV (lgnoffo et al., 1995). In contrast, disruption ofpep gene expression in LdMNPV by partial deletion or insertion of the coding sequence for green fluorescent protein (GFP) did not result in any significant difference in virulence against the gypsy moth, Lymantria dispar (Bischoff & Slavicek, 1999). In this paper, we describe the construction ofpep-mutant RoMNPV, and biological properties of the mutant viruses.

MATERIALS AND METHODS

Insects. Eggs of Ostrinia nubilalis were obtained from the USDA-ARS Com Insects & Crop Genetics Research Unit, Ames, IA. Trichoplusia ni eggs were obtained from Dr. Tom Coudron, USDA-ARS, Columbia, MO. Heliothis virescens eggs were obtained from Southland Products, Lake Village, AR. All insects were reared at 27°C with a photoperiod of 12 hours light and 12 hours dark. 0. nubilalis was reared on wheat germ diet (Guthrie, 1987). T. ni and H. virescens were reared on diets from Southland Products, Lake Village, AR.

Cells and viruses. S:f9 cells (cloned from the Spodoptera frugiperda IPLB-Sf21-AE cell line: Vaughn et al., 1977) were grown in TNM-FH medium (JRH Biosciences, Lexena, KS) supplemented with 3% heat-inactivated fetal bovine serum (Intergen company, NY), 1% streptomycin/ penicillin (Sigma), and 0.01 % (w/v) pluronic F-68 (JRH Biosciences, Lexena, KS). The AcMNPV clone C6 (Possee, 1986) and RoMNPV clone Rl (Smith & Summers, 1980) were used as viruses.

Antibodies. A polyclonal rabbit antiserum raised against Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (OpMNPV) PEP was obtained from Dr. George F. Rohrmann, Oregon State University, Corvallis, OR (Gombart et al., 1989). Goat anti-rabbit- 17

horseradish peroxidase (Sigma) was used as secondary antibody for western blot analysis. Goat anti-rabbit antibody conjugated with 10 run gold particles (Ted Pella Inc., Redding, CA) was used as secondary antibody for immunogold electron microscopy.

Alignment and phylogenetic analyses ofthe RoMNPVpep gene with homologs in other baculovirus genomes. The RoMNPV pep gene was sequenced by Diane Schroeder and Dr. Robert Harrison, Iowa State University, Ames, IA. A BLAST search was carried out to find homologous ORFs (Altschul et al., 1990; Altschul et al., 1997). Multiple aligrunents of 10 polyhedral envelope protein predicted animo acid sequences were performed using the CLUSTALW method with the program DNAStar (Lasergene) (Thompson et al., 1994). The aligrunent file was analysed using neighbor joining in PAUP* 4.0b 10 (Swofford, 2000) to draw a phylogenetic tree.

Construction ofthe recombinant virus RoPEPLacZ. The location of the RoMNPV pep gene was identified by Southern blot analysis using an AcMNPV restriction fragment containing pep as probe (Zuidema et al., 1989). Restriction fragments that hybridized with the probe were cloned and the RoMNPV pep gene was sequenced. The RoMNPV 7 .6 kb HindIII-G fragment that contains the entire pep ORF was cloned into pGEM-9zfto produce pRoHindIII-G. The 5' end ofpep was removed by restriction digestion with Xbal and Bg!II, leaving the 2.6 kb BglII-HindIII fragment in the vector. The lacZ gene under control of an hsp70 promoter (Zuidema et al., 1989) cut withXbaI and BamHI was inserted into pRoHindIII-G/XbalI-BglII. Then the 4.0 kb Xbal fragment containing the partial pep gene was inserted upstream of the lacZ gene. The recombinant plasmid was named pRoPEPLacZ. S:£9 cells were cotransfected with RoMNPV-Rl viral DNA and pRoPEPLacZ DNA using calcium phosphate precipitation (Smith et al., 1983) or lipofectin (King & Possee, 1992). After 7 days, an aliquot of medium from the cotransfection culture was used for a plaque assay. Plaque assays were stained after 5-6 days with 200µ1 staining solution [0.25% neutral red, 0.25%X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)] in PBS buffer

(137mM NaCl, 2.7mM KCl, lOmM Na2HP04, 2mM KH2P04, pH 7.4) per petri dish. Plaques of recombinant virus (named RoPEPLacZ) were identified by blue staining against a 18

white background (Summers & Smith, 1987). Three rounds of plaque purification were conducted. Viral DNA was extracted from budded virus in infected cell culture medium. An equal volume of20% P.E.G.8000 (polyethylene glycol 8000)/ lM NaCl was added to the medium, mixed and incubated overnight on ice. Budded viruses were pelleted by centrifugation at 5,000g for 15 min at 4°C (Summers & Smith, 1987). One ml DNAzol reagent (GIBCO BRL) was added to dissolve the pellet, and 0.5ml 100% ethanol was added to precipitate the viral DNA. The mixture was incubated for 3 min at room temperature and centrifuged at 4,000g for 2 min. The supernatant was removed, and 95% ethanol was added to wash the DNA pellet. One hundred µ.l of 8 mM NaOH was added to dissolve the pellet, and O.lM HEPEP [4(-2-hydroxyethyl)-1-piperazineethanesulfonic acid] was added to adjust the pH to 7.5 once the pellet had completely dissolved. Extracted viral DNA (-5µ.g) was examined by restriction enzyme analysis to confirm that the expected recombination had occurred.

Site-directed mutagenesis ofthe pep gene and construction ofa series ofpep- mutant RoMNPV. The Transfer site-directed mutagenesis kit (Clontech) was used to eliminate the start codon ATG ofpep (ATG - ATC), and insert a stop codon TAG (TCG -TAG) 37 nucleotides (nt) downstream of the start codon (Fig. 1) according to the manufacturer's direction. Four primers were used for mutagenesis: the selection primer (named PEP-STOP, 5' -CCAGTGATATCGAATTCGGTACCCGGG-3 ') replaced the SacI site with an Eco RI site, the mutagenic primer 1 (named PEP, 5 ' - CAATATTTCAAAATATCAAACCGACGAATAAC-3 ')replaced the initiation ATG with ATC, the mutagenic primer 2 (named STOP-PEP 5' - TTCGACGACCGCTAGGTCCTTTGGATC-3') replaced the codon TCG for serine with TAG, and the bridge primer (named pp342, 5'-CTCCTCATTGCAGACCTC-3') bound +335-353 nt downstream from the start coden. Plasmids containing the pep-mutants, pRoPEP f).ATG, pRoPEPTAG, and pRoPEP f).ATG-TAG, were produced with one or both of these mutations. Sf9 cells were cotransfected with Bsu361 linearized RoPEPLacZ parental virus DNA and the plasmids containing the pep-mutants (Sambrook & Russell, 2001) using calcium phosphate precipitation (Summers & Smith, 1987). Plaques of the three pep-mutant 19

viruses, RoPEP/1ATG, RoPEPTAG, and RoPEP/1ATG-TAG were identified as white plaques against a blue plaque background. Plaque purification, viral DNA extraction and restriction were conducted as described above. Three restriction enzymes, HindIII, EcoRI or Pstl, were used for digestion. After digestion for 3 hours in a 37°C water bath, DNA samples were loaded onto a 20 X 25 cm 0.8% agarose gel and were electrophoresed for 14 hours at 75 v.

PCR and nucleotide sequencing. The mutated pep gene in each recombinant virus was amplified by PCR. DNA from the viruses RoPEP /1ATG, RoPEPTAG, and RoPEP/1ATG-TAG was extracted as described above and used as templates in PCR reactions. The primers pp341 (5'-GAACGTGGGATGCTGTTG-3') and pp2 (5'- CGACGACGGTAAAAGTAG-3') were used for amplification of the 5' end of the mutated pep genes. Native pfu taq polymerase (Strategene) was used to amplify the target fragment. PCR products were purified using Microcon 100 filters (Amicon) and were sequenced using a 96-well Applied Biosystems 377 automated DNA sequencing system by the DNA Sequencing and Synthesis Facility at Iowa State University.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA GE) and Coomassie brilliant blue staining. Sf9 cells were seeded in 60-mm petri dishes (2.5Xl 06 cells/dish) and infected with AcMNPV-C6, RoMNPV-Rl, RoPEP/1ATG, RoPEPTAG, RoPEP/1ATG-TAG and RoPEPLacZ at a multiplicity of infection (MOI) of 1. One dish of uninfected Sf9 cells was used as a control. Cells were harvested 72 hours post-infection, washed with phosphate buffered saline (PBS, 137mM NaCl, 2.7mM KCl, lOmM Na2HP04,

2mM KH2P04, pH 7.4) and resuspended in lX SDS-PAGE disruption buffer (Sambrook & Russell, 2001). The cell lysates were sheared by using 27-gauge needles, and then boiled for 5 min at 100°C. Fifty thousand cell equivalents per sample were loaded on 12% SDS-P AGE (16xl8 cm) gel (Laemmli, 1970). The gel was stained with Coomassie blue R-250 staining solution, destained in 40% (v/v) methanol, 10% (v/v) glacial acetic acid in ddH20 (Sambrook & Russell, 2001) and dried using Model 583 gel dryer (BIO-RAD). SDS-PAGE analysis of protein samples was repeated three times. 20

Western blot analysis. The proteins separated on 12% SDS-PAGE gels were transferred to Hybond-P PVDF membrane (Amersham Life Science, Arlington Heights, IL) for western blot analysis. The membrane was blocked with blocking buffer (5% dry milk, 0.1 % Tween-20 in Tris buffered saline, pH 7 .6), then incubated with a 1 :2000 dilution of the anti-OpMNPV PEP polyclonal antiserum for one hour at room temperature. The membrane was washed twice with washing buffer (1 % dry milk, 0.1 % Tween-20 in Tris buffered saline, pH 7 .6), and then incubated with a dilution of 1 :2000 goat anti-rabbit- horseradish peroxidase for 1 hour at room temperature. Bound antibodies were visualized using the Enhanced Chemiluminescence western blotting detection reagents (Amersham Life Science).

Amplification and purification ofpolyhedrafrom insects. Stocks ofRoMNPV-Rl, RoPEP!:iATG, RoPEPTAG, RoPEP!:iATG-TAG and RoPEPLacZ were produced in fifth instar larvae of H. virescens. Larvae were infected with polyhedra produced in vitro in Sf9 cells. Polyhedra concentrations were determined by using a phase contrast microscope and a Neubauer bright-line hemocytometer (Fisher Scientific, Pittsburg, PA). Fourth instar H. virescens larvae were placed individually in 5/8 oz plastic cups (Fill-Rite Inc., Newark,

NJ) without diet and incubated at 27°C overnight. Individual cubes of diet (- 4 mm3) inoculated with 500,000 polyhedra ofRoMNPV or pep-mutant viruses were then added to each cup. Larvae that completely consumed the inoculated diet cube were then supplied with clean diet and incubated at 27°C. Once larvae had died approximately six days post- infection, cadavers were collected and homogenized in 0.1 % SDS (1 ml per cadaver, 10-15 cadavers at a time) using a 30 ml dounce homogenizer (Wheaton Scientific Products Inc., Millville, NJ). The pestle was moved up and down at least 10 times in the dounce, with the pestle remaining submerged. The homogenates were filtered through 5 layers of cheesecloth, and the dark brown eluents containing the polyhedra were transferred to 42 ml Pyrex glass tubes and pelleted at 3,600g for 10 min at 4°C. The pellets were resuspended in 15 ml 0.5% SDS, centrifuged at 3,600g for 10 min at 4°C and resuspended in 15 ml 0.5M NaCl, centrifuged at 3,600g for 10 min at 4°C (O'Reilly et al., 1992). The pellets of polyhedra were 21

resuspended in a small volume(~ 1 ml) of distilled water, and sodium azide added to a final concentration of 0.02% to prevent bacterial growth. Virus stocks were stored at 4°C.

Scanning electron microscopy. Polyhedra isolated from H. virescens cadavers were dehydrated in 70% and 100% ethanol for 5 min, respectively. After air-drying, ~2 x 106 polyhedra of each virus were placed on aluminum stubs, sputter coated with a thin layer of gold I palladium and examined on a JEOL 5800 LV SEM at 1OkV (Bischoff & Slavicek, 1999; Bozzola & Russell, 1999).

Transmission electron microscopy. Sf9 cells were seeded in 75 cm2 flasks and infected with RoMNPV-Rl, RoPEP!:iATG, RoPEPTAG, RoPEP!:iATG-TAG or RoPEPLacZ at a MOI of 5. Cells were harvested at 72 hours post-infection, pelleted at 1000 g for 10 min and fixed by incubating in 1% glutaraldehyde in PBS overnight at 4°C. The cells were washed three times with PBS, and post-fixed in 1% osmium tetroxide in PBS for 1 hour at room temperature. The cells were washed twice with PBS and once with distilled water. Cells were then dehydrated by incubation in a series of ethanol concentrations (25%, 50%, 70%, 80%, 85%, 90%, 95%, and three times in 100%) and cleared with three washes in 100% acetone. Each incubation lasted 15 min. The samples were infiltrated with acetone: EMBED 812 epoxy resin (Electron Microscopy Sciences, Fort Washington, PA) at ratios of 3:1, 1:1, and 1 :3, followed by pure resin, for 12 hours for each step. The cells were resuspended in fresh resin and transferred to BEEM (Better Equipment for Electron Microscopy) capsules (Electron Microscopy Sciences). Resin was polymerized for forty- eight hours at 65°C. Sectioning of embedded samples was carried out on a Reichert Ultracut S ultramicrotome using a diatome diamond knife. Thin sections were collected on formvar- coated nickel grids. Grids were stained with 4% uranyl acetate in 50% ethanol for 20 min, followed by Sato-S lead acetate for 15 min, and were examined on a Jeol 1200 EX scanning/transmission electron microscope at 80 KV.

Immunogold electron microscopy. Sf9 cells were infected with RoMNPV-Rl, RoPEP!:iATG, RoPEPTAG, RoPEP!:iATG-TAG or RoPEPLacZ at an MOI of 5 and 22

harvested at 96 hours post-infection. The cells were pelleted and fixed in 1% paraformaldehyde-0.5% glutaraldehyde-0.05M sodium cacodylate, pH 7.1, for 10 min at 4°C, then in 2% paraformaldehyde-2.5% glutaraldehyde-0.05M sodium cacodylate, pH 7.1, for 30 min at 4°C. After washing the cells three times with 0.05M sodium cacodylate, the cells were dehydrated with a series of ethanol concentrations (30%, 50%, 70%, 90% and three times with 100%) for 30 min at each step at 4°C. Cells were then infiltrated with ethanol: LR White Resin (London Resin Company Ltd, England) using ratios of 1: 1, and 1 :3 followed by pure LR White (1 hour for each step), and then incubated twice with pure resin for 12 hours at 4°C. The cells were resuspended in LR White Resin and transferred to gelatin capsules for embedment. Resin was polymerized at 50°C for 48 hours (Vandenbosch, 1991). Gold labeling of thin sections was carried out (Erickson et al., 1993). Grids were first immersed in 25 µJ drops of TBS-supplemented buffer (0.05M Tris, 0.85% NaCl, 0.5% normal goat serum and 0.5% BSA, pH 8.3-8.5) with 3% non-fat dry milk for 2 hours at room temperature. Grids were then immersed in drops of 1: 100 primary antiserum diluted in TBS- supplemented buffer with 3% non-fat dry milk for 2 hours at room temperature. After stream washing of grids followed by an additional three washes in drops of TBS-supplemented buffer, the grids were immersed in a drop of a 1: 100 dilution of secondary antibody conjugated with 1Onm gold particles for 2 hours at room temperature. After stream washing and three washes in drops of distilled water, grids were stained with 2% aqueous uranyl acetate for 10 min and examined on a Jeol 1200 EX scanning/transmission electron microscope at 80 KV.

Analysis ofthe sensitivity ofpolyhedra to alkali. Polyhedra isolated from H. virescens cadavers were diluted in distilled water to a concentration of 8x 104 polyhedra per µJ and placed on carbon-coated form.var copper grids. After air-drying, grids were incubated in weak alkali (0.05M Na2C03, pHl0.9) for 0, 1, 2, and 10 min, stained with 2% potassium phosphotungstic acid (pH6.5), and examined for release of occlusion-derived virions on a JEOL 1200 EX scanning/transmission electron microscope at 80 kV.

Bioassays. Lethal concentration bioassays were carried out with the RoMNPV-Rl, RoPEP!iATG, RoPEPTAG, and RoPEP!iATG-TAG virus stocks using the droplet feeding 23

technique (Hughes et al., 1986) with first instar 0. nubilalis and T ni, respectively. First instar larvae were allowed to drink from one of five concentrations of polyhedral occlusion bodies (POB): 1,000, 5,000, 25,000, 125,000, 625,000 POB/µl for 0. nubilalis; 4, 20, 100, 500, 2,500 POB/µl for T ni. Each virus solution contained 4% blue food coloring (McCormick & Co. Inc.). Controls were provided with 4% blue food coloring in distilled water alone. Larvae that had ingested virus suspension were identified by blue coloration of the gut, and transferred individually to containers with artificial diet by using paintbrushes. Larvae were maintained at 27°C until pupation or death. Thirty-five larvae were used per dose. Larvae were checked for handling deaths and escapes after 48 hours, and mortality was scored twenty-one days post-infection for 0. nubilalis or seven days post-infection for T ni. Bioassays were replicated three times. Data were subjected to probit analysis (Russell et al., 1977) and the assumptions of the models verified (Robertson & Preisler, 1992).

Analysis ofmidgut pH. To determine the midgut pHs, first instar 0. nubilalis and T ni were fed with broad range pH indicator solutions: 0.1 % neutral red (pH 6.8-8.0), 0.01 % phenol red (pH 6.8-8.4), 0.05% meta-cresol purple (pH 7.4-9.0), 0.05% thymol blue (pH 8.0- 9.6), 0.2% aniline blue (pH 10.0-13.0), 0.1 % acid fushin (pH 12.0-14.0), 0.5% carmine indigo (pH 11.6-14.0). pH indicator dyes were dissolved in distilled water. The gut coloration of the larvae was observed by using a dissection microscope. Thirty larvae were tested for each indicator dye, and the experiments were replicated three times (Castillejos et al., 2001).

Quantification ofocclusion-derived virions per polyhedral unit area. Polyhedra of

RoMNPV-Rl, RoPEPLacZ, RoPEP~ATG, RoPEPTAG, and RoPEP~ATG-TAGwere fixed, embedded, sectioned and stained as described above for transmission electron microscopy. The numbers ofvirions were counted in 30-41 polyhedral cross-sections selected randomly from grids of each virus. The areas of each polyhedral cross-section were determined by using the Scion Image program. One-way analysis of variance (ANOVA), Tukey' s and Bonferroni tests were used to analyze the number of virions per µm2 polyhedral cross-sectional area by SAS program (SAS Institute Inc., Cary, NC). 24

Nucleocapsids per occlusion-derived virion. Insect-derived polyhedra (~2.0x109

polyhedra) were incubated in 1 ml dilute alkaline saline (O. lM Na2C03, 0.05M NaCl, pHl0.9) for two hours at 37°C to dissolve the polyhedrin matrix and release the occluded virions. Samples were checked under the light microscope to ensure that polyhedra were solubilized. Sucrose gradients (25-56% sucrose w/v in I lOmM Tris pH7.5) were made in SW41 centrifuge tubes (Beckman, Palo Alto, CA) by using a Model 107-201M gradient master (BioComp, New Brunswick, Canada). One ml samples ofvirions were overlaid carefully onto the 1 lml 25-56% continuous sucrose gradients and centrifuged at 53,000g for 30 min at 10°C using an SW41 rotor (Beckman). Photographs were taken of the centrifuge tubes by using a digital camera. The intensities of the bands in these images were determined by using the Scion Image program (Scion Corporation, Frederick, MD).

RESULTS

Comparison ofbaculovirus pep genes. The polyhedral envelope protein I calyx amino acid sequences of the following ten baculoviruses were aligned using the ClustalW method (Fig. 2): RoMNPV, AcMNPV, BmMNPV, CfMNPV, EppoMNPV, OpMNPV, HaSNPV, LdMNPV, SeMNPV, and SpltMNPV. AcMNPV ORF131 showed the highest identity to RoMNPV PEP (99.4%). OpMNPV ORF129 whose polyclonal antiserum was used for examination of RoMNPV PEP by western blotting and immunogold-EM showed 65% identity with RoMNPV PEP. The unrooted phylogenetic tree (Fig. 3) made by neighbor joining showed that the pep genes segregated into the same NPV groups (I and II) as has previously been described (Hemiou et al., 2001). Group I included RoMNPV, AcMNPV, BmMNPV, C£NPV, EppoMNPV and OpMNPV; group II included LdMNPV, HaSNPV, SpltMNPV and SeMNPV. Group I is more conserved than group II, with group II being highly divergent.

Examination ofrecombinant virus genomes. Restriction enzyme digestion was used to determine whether a double homologous recombination event occurred between the 25

parental virus, RoPEPLacZ, and the plasmids, pRoPEP!iATG, pRoPEPTAG or pRoPEP !iATG TAG, to introduce the mutant pep genes into the virus genome. Following digestion with HindIII, RoPEP !iATG, RoPEPTAG, and RoPEP !iATG-TAG showed the same DNA fragment pattern as RoMNPV Rl, which was distinct from that ofRoPEPLacZ (Fig. 4). Specifically, HindIII digestion of the RoPEPLacZ genome resulted in two additional fragments of 5.4 and 5.9 kb, and loss of the 7.6. kb HindIII-G fragment produced on digestion of the other virus genomes (Fig. 4). The restriction enzymes, EcoRI and Pstl, were also used for analysis of the virus genomes (data not shown). Restriction enzyme analysis confirmed the expected genome structure of the pep-mutant viruses. The mutated pep genes were sequenced at the Iowa State University DNA Synthesis and Sequencing Facility. The sequencing results confirmed the elimination of the start codon

ofpep (ATG ~ATC) in RoPEP!iATG, the presence of the inserted stop codon TAG (TCG

~TAG) 37 nucleotides (nt) downstream of the start codon in RoPEPTAG, and the presence of both mutations in RoPEP!iATG-TAG.

Analysis ofPEP expression by wild-type and recombinant RoMNPV. To determine whether PEP was expressed by the pep-mutant viruses, virus infected Sf9 cells were harvested at 72 hours post-infection, proteins were separated by 12% SDS-PAGE and visualized by staining with Coomassie blue R-250 (Fig. 5A) or by western blot (Fig. 5B). The strong band of approximately 30 kDa present in virus-infected cells but absent from the mock infected Sf9 cells after Commassie blue staining, is polyhedrin, the highly expressed major constituent of polyhedra. PEP bands are hard to detect from the Commassie blue- stained gel. AcMNPV PEP (pp34) with 322 amino acids, and RoMNPV PEP with 321 amino acids have similar predicted sizes of ~36 kDa. RoMNPV PEP shares 99.4% identity with AcMNPV PEP, while OpMNPV PEP shares 66.4% amino acid identity with RoMNPV PEP. The anti-OpMNPV PEP antiserum recognizes both OpMNPV PEP and AcMNPV PEP (Lent et al., 1990). Western blot results showed strong expression of PEP in Sf9 cells infected with AcMNPV, RoMNPV and RoPEP!iATG, and greatly reduced PEP expression in Sf9 cells infected with RoPEPTAG or RoPEP!iATG-TAG (Fig. 5B). Cross-reactive proteins in RoPEPTAG or Ro PEP !iATG-TAG- infected cells were also smaller in size (29 kDa 26

compared to approximately 34 kDa for RoMNPV Rl). No PEP was detected in Sf9 cells infected with RoPEPLacZ (Fig. 5B). Some non-specific cross-reactivity was detected in each lane, including mock. Bands ofless than 30 kDa detected in the AcMNPV, RoMNPV and Ro PEP /1ATG lanes, may be PEP degradation products.

Analysis ofpolyhedra by scanning electron microscopy. Analysis of the surface of polyhedra by scanning electron microscopy revealed that all of the pep-mutant viruses (RoPEP/1ATG, RoPEPTAG, RoPEP/1ATG-TAG and RoPEPLacZ) had pitted surfaces, in contrast to the smooth appearance ofRoMNPV (Fig. 6). There was no obvious difference in size between the polyhedra of pep-mutant and wild-type viruses.

Polyhedral envelope morphogenesis and PEP localization. Analysis of the polyhedral envelopes 72 hours post infection by transmission electron microscopy showed the presence of an electron dense polyhedral envelope surrounding RoMNPV polyhedra (Fig. 7A). The sharp, clearly defined envelope seen for RoMNPV polyhedra was absent from the polyhedra of the pep-mutant viruses (Fig. 7B-E), although it is unclear whether an envelope was present on polyhedra ofRoPEP/1ATG (Fig. 7B). To determine the location of PEP in virus infected Sf9 cells by immunogold electron microscopy, cells were harvested at 96 hours post-infection. Immunogold electron microscopy revealed specific labeling on the periphery of polyhedra ofRoMNPV (Fig. 8A) and RoPEP /1ATG (Fig. 8B), but no specific labeling on the polyhedra of RoPEPTAG (Fig. 8C), RoPEP/1ATG-TAG (Fig. 8D) and RoPEPLacZ (Fig. 8E). Control grids were set up for each reaction, and were only incubated with secondary antibody (goat-anti-rabbit conjugated with 1Onm gold). No specific labeling was detected on control grids (data not shown).

Sensitivity ofpolyhedra to alkali. Infectious virions are embedded in a crystalline matrix of polyhedrin and released when polyhedrin dissolves in the alkaline environment of the insect midgut. To determine whether the polyhedra of pep-mutant RoMNPV are more easily dissolved than those of wild-type RoMNPV, polyhedra were incubated in 0.05M Na2C03 (pH 10.9). On incubation in alkali, RoMNPV polyhedra became swollen with 27

decreased electron density, but retained their integrity without apparent release of virions after a 10 min incubation period (Fig. 9, row A). In contrast, polyhedra of the pep-mutant RoMNPV dissolved quickly, allowing the release of virions after a 2 min incubation period (Fig.9, rows B-E). Because of variation between polyhedra of each virus, it was not possible to determine whether there were significant differences in alkaline sensitivity among the pep- mutants.

Lethal concentration bioassays. Lethal concentration bioassays were carried out with the pep-mutant viruses in a semi-permissive host 0. nubilalis and in a permissive host

T ni (Table 2). RoMNPV had a significantly lower LC50 compared to those of the pep-

mutant viruses RoPEP~ATG, RoPEPTAG and RoPEP~ATG-TAG in 0. nubilalis. There were no statistically significant differences in virulence between pep-mutant RoMNPV and wild-type RoMNPV in the permissive host, T ni (Table 2).

Midgut pH of 0. nubilalis and T. ni. Differences in midgut pH between 0. nubilalis and T ni could account for the different response of these species to pep-mutants in bioassays. To determine the midgut pH of 0. nubilalis and T ni larvae, first instars were fed with one of seven pH-indicator solutions covering a pH range of 6.8-14.0. It was not possible to determine whether larvae consumed neutral red (with a pH range of 6.8-red to 8.0-yellow) because a color change was not noticed. Larvae refused to consume thymol blue. The larvae did consume phenol red (6.8 yellow-8.4 red) with the gut appearing red, meta-cresol purple (7.4 yellow-9.0 purple) with the gut appearing purple, carmine indigo (11.6 blue-14.0 yellow) with the gut appearing blue, acid fushin (12.0 red-14.0 colorless) with the gut appearing red, aniline blue (10.0 blue-13.0 carmine) with the gut appearing blue (Fig. 10). There was no obvious difference in the midgut coloration between the two species, with a pH of between 9.0 and 11.0 in both cases.

Quantification ofoccluded virions. The numbers ofvirions occluded within polyhedra of each virus type are presented as the average number ofvirions per µm2 of polyhedral cross-sectional area (Fig. 11). Polyhedral cross-sections (30 - 41 per treatment) 28

were randomly selected from transmission electron micrographs for each virus. RoMNPV polyhedra with 12 ± 3.8 virions per µm2 had significantly more virions than pep-mutant RoMNPV with from 7 ± 2.4 virions per µm2 to 8 ± 2.1 virions per µm2 (ANOVA, Tukey test, Bonferroni, p<0.05). There was no significant difference between the numbers of virions per µm2 of polyhedral cross-sectional areas for RoPEP~ATG, RoPEPTAG, RoPEP~ATG- TAG and RoPEPLacZ (Fig. 11 ).

Nucleocapsids per virion. Nucleocapsids may be enveloped singly or in bundles within the envelope of the occlusion-derived virion. Packaging of multiple nucleocapsids within the virion may result in increased virulence (Washburn et al., 1999). Ultracentrifugcation within a continuous sucrose gradient was used to separate virions containing different numbers ofnucleocapsids for each virus (Fig. 12). Each white band in the sucrose gradient tube represents purified virions containing the same number of nucleocapsids, from one nucleocapsid per virion (highest band) to - 9 nucleocapsids per virion (lowest band). Preliminary quantification of ODV bands was performed with Scion image. The areas of each peak were determined and the ratios of virions containing different number ofnucleocapsids were calculated (Fig. 12; Table 3). From these preliminary data, we conclude that there were no major differences in the numbers of nucleocapsids packaged per virion between the viruses.

DISCUSSION

Phylogenetic analysis ofpep genes from ten baculoviruses confirmed the relatedness between RoMNPV and AcMNPV among the group I baculoviruses (Zanotto et al., 1993). Baculovirus phylogenetic analysis has been conducted by alignment primarily ofpolyhedrin gene sequences (Zanotto et al., 1993), although different phylogenetic trees result from analysis of different genes (Federici & Hice, 1997). There are 63 genes that are common to the 9 baculoviruses sequenced to date (Hyink et al., 2002) and phylogenetic consideration of all common genes may provide a better indication ofbaculovirus relatedness and origin. 29

However, given the ability ofbaculoviruses that infect common hosts to recombine (Crozier et al., 1994; Kondo & Maeda, 1991) and the hypothetical transfer of genes to and from insect hosts and other pathogens (Herniou et al., 2001), results from phylogenetic analysis of multiple baculovirus genes should be interpreted with caution. In the absence of an outgroup, an unrooted tree was constructed. Rather than disrupting pep by insertion of a foreign gene, or by partial deletion of the pep gene, we used site-directed mutagenesis to disrupt PEP expression. This approach was taken because site-directed mutagenesis is less likely to affect transcription of other ORFs near to the pep gene.

Western blot results showed that RoPEP~ATG produced wild-type levels of PEP, yet the polyhedral envelope appeared to be missing on examination by scanning electron microscopy. This suggests that, if translation for this mutant pep gene begins at the next ATG that is seven codons downstream from the first ATG, then these first seven amino acids contain a domain required for formation of a polyhedral envelope with a normal level of

structural stability. N-terminal sequencing of the PEP produced by RoPEP~ATG or peptide mapping by protease digestion could be performed to determine where translation is initiated. We also tested the hypothesis that PEP N-terminal sequences play a role in nuclear localization by examining nuclear and cytosolic fractions for the presence of PEP. Western

blot analysis showed that PEP produced by RoPEP ~ATG was present only in the nucleus (data not shown).

For RoPEPTAG and RoPEP ~ATG-TAG infected Sf9 cells, cross-reactive bands of lower molecular mass (around 29 kDa) were detected indicating that translation occurred past the inserted stop codon. There are 10 in-frame A TGs in the pep gene sequence, and the third in-frame ATG is 28 codons downstream from the first ATG. There is an A residue at the -3 position relative to the A of this ATG, which conforms to Kozak's rule (Kozak, 1986; Kozak, 1987) and data for translation initiation for other baculovirus genes (Ayres et al., 1994). Hence the bands may result from translation initiation at the third in-frame ATG. These truncated proteins were expressed at lower levels compared to RoMNPV PEP, and did not produce a polyhedral envelope. Electron microscopy results confirmed the absence of the polyhedral envelope on the surface of the polyhedra ofRoPEPTAG and RoPEP~ATG-TAG. 30

One possible explanation for the low expression of truncated PEP is that the short ORF (minicistron) created upstream (39 nt and 21 nt, respectively) reduced the efficiency of translation initiation from the third in-frame ATG in RoPEPTAG and RoPEP!iATG-TAG. Upstream short ORFs (minicistrons) have been reported for other baculovirus genes (Guarino & Smith, 1990; Lu & Carstens, 1992; Wu & Miller, 1989). Mutagenesis of a conserved minicistron upstream ofbaculovirus gp64 confirmed that the minicistron functions to negatively regulate gp64 translation efficiency (Chang & Blissard, 1997). Interestingly, there also appears to be a minor 29 kDa band in AcMNPV-C6 and RoPEP !iATG lanes (Fig. SB), which suggests that translation initiation may naturally occur at downstream ATGs. The polyhedra ofpep-mutant RoMNPV have a pitted surface in contrast to the smooth appearance of wild-type virus in scanning electron micrographs (Fig. 6). Similar observations were reported for the pep-mutants of OpMNPV (Gross, 1994) and LdMNPV (Bischoff & Slavicek, 1999). The rod-shaped pits on the pep-mutant viruses may result from loss of virions from the periphery of the polyhedra. The pitted surface is consistent with the absence of the complete polyhedral envelope. Immunogold labeling of the RoPEP!iATG polyhedra suggests that PEP is distributed on the surface of polyhedra (Fig. 8B). RoPEP!iATG may have a weak, incomplete polyhedral envelope that is easily disrupted. Reduced stability of the envelope ofRoPEP!iATG would explain why RoPEP!iATG polyhedra dissolve more readily in alkali (Fig. 9B). The other pep-mutant RoMNPV, namely RoPEPTAG, RoPEP!iATG-TAG and RoPEPLacZ, show no specific labeling in immuno- gold electron micro graphs, which is consistent with the pitted polyhedral surface seen in scanning electron micrographs. Alkali sensitivity experiments showed that polyhedra ofpep-mutant RoMNPV were more readily dissolved allowing more rapid release of virions than wild-type virus in O.OSM

Na2C03 (pHl 0.9). Similar observations were reported for the AcMNPV pep-mutant AcDZS PEP- (Zuidema et al., 1989). Ignoffo et al. reported that the virulence of AcDZS PEP- was enhanced six-fold compared to AcMNPV against first instar T. ni because of the increased sensitivity to alkali (Ignoffo et al., 1995). However, our bioassay results showed the pep- mutant viruses had reduced virulence relative to RoMNPV against a semi-permissive host, 31

the European com borer 0. nubilalis, and unaltered virulence against a permissive host, the cabbage looper T. ni. Although the RoPEPlacZ was used as a negative control virus for protein studies, this virus was not used as a negative control in insect bioassays. The reason for this is that recombinant baculoviruses expressing p-galactosidase have previously been shown to have unexpected biological activity that may affect survival time or lethal dose (Hughes et al., 1997; O'Reilly et al., 1990; Passarelli & Miller, 1994; Wood et al., 1993). The reason for the alteration in virus properties caused by p-galactosidase is unknown. This enzyme is present within lysosomes in athrocytes and various other lepidopteran tissues. One hypothesis for the different effects of PEP deletion on the semi-permissive and permissive host insects, and indeed to the different susceptibilities of T. ni and 0. nubilalis to baculovirus infection, relates to the midgut pH. An alkaline pH is required for efficient dissolution of polyhedra and release of infectious virions. Determination of the midgut pHs of first instar 0. nubilalis and T.ni by using pH-indicator solutions showed no obvious differences in the midgut pH between the two species. Based on our observations, we conclude that the midgut pH of first instar larvae of both 0. nubilalis and T.ni is between 9.0- 11.0, which differs from previous reports (Berenbaum, 1980; Raun et al., 1966). There are several factors that may contribute to the differences in recorded pH values including (1) variation in pH of the gut at different larval stages (we tested neonate larvae, while previous reports are for late instar larvae), (2) variation in the type of food consumed that can affect the gut pH (Keating et al., 1990), and (3) differing sensitivity of techniques used to record pH. The pH indicator solutions have a relatively broad pH range. Other more precise methods, such as self-referencing ion-selective liquid ion exchanger (SERIS-LIX) microelectrode techniques (Boudko et al., 2001; Smith et al., 1999), can be used to provide more accurate pH measurements within the insect midgut lumen. However, the ability of polyhedra to dissolve is not dependant on pH alone but also varies with ionic strength (Kawanishi & Paschke, 1970). Another hypothesis to explain the difference in virulence against 0. nubilalis and T. ni is that more rapid release of virions from polyhedra of the pep-mutant viruses within the insect midgut results in longer exposure of virions to degradative enzymes or other factors in 32 the gut of 0. nubilalis. Occlusion derived virions are susceptible to degradation by midgut enzymes in some species (Granados & Williams, 1986). Hence, degradation ofvirions within the gut of 0. nubilalis may also be a contributing factor to the reduction in virulence seen for this species. Differences in factors present in 0. nubilalis and T ni guts may contribute to the different bioassay results seen for these two species. This hypothesis could be tested by incubating ODV ofRoMNPV with midgut extracts from 0. nubilalis and T. ni, and testing for viability of the ODV by bioassay in T ni. It appeared from some of the electron micrographs that some of the pep-mutant viruses had fewer virions occluded per polyhedron (see figure 9D for example). Quantification of occluded virions within polyhedra of each virus type provided a possible explanation for the bioassay results. Fewer virions were occluded within the polyhedra of pep-mutant viruses. Statistical analysis showed that pep-mutant RoMNPV had significantly fewer virions (7-8 per µm2 of polyhedral cross-sectional area) than RoMNPV (12 per µm2 of polyhedral cross-sectional area). Virions may have been lost from the pep-mutant polyhedra by becoming dislodged from the periphery of the polyhedra. In bioassays, test insects of the same species consumed the same numbers of polyhedra, but fewer virions would have been released by pep-mutant RoMNPV in the insect midgut compared to the virus. The reduction in the numbers of virions released from the pep-mutant viruses may have resulted in a decrease in virulence against the semi-permissive host, but not in the permissive host T. ni. T. ni is very susceptible to infection by RoMNPV, with an LC so of 88 OBs/µl (Table 2). Neonate T.ni were reported to ingest a volume of 5.83±0.40 nl in droplet feeding experiments

(Kunimi & Fuxa, 1996). Hence the LD50 ofRoMNPV is around 0.51 OB. Given the high susceptibility of T.ni, a detectable drop in virulence would not be expected from a 33% reduction in the numbers ofvirions per polyhedron under the bioassay conditions used. In contrast, the reduction in the numbers of occluded virions resulted in a significant loss of virulence against 0. nubilalis, which might be expected given the high LC50 (6611 OBs/µl, Table 2) of RoMNPV against this species. Neonate 0. nubilalis ingest around 4.0 nl (Dr. L.

C. Lewis, unpublished) and the LD50 for RoMNPV is around 2.64 OBs. An alternative or complementary hypothesis is that midgut factors present in 0. nubilalis but absent from T. ni 33

degrade virions, thereby contributing to the drop in virulence of pep-mutant viruses in this species. In addition to fewer virions being occluded per polyhedron, envelopment of fewer nucleocapsids per occlusion-derived virion could also result in reduced virulence (Washburn et al., 1999). We conducted preliminary experiments to examine the numbers of nucleocapsids per virion to determine whether there were any major differences between the pep-mutant viruses and RoMNPV. Virions containing different numbers ofnucleocapsids were released from polyhedra by alkaline lysis, and separated on continuous sucrose gradients. Bands ofvirions with different numbers of nucleocapsids per virion were clearly visible in the gradients. Although the same numbers of polyhedra of each virus were used for alkaline lysis, the virion bands were consistently stronger for the pep-mutant viruses compared to the virus. This observation supports the hypothesis that pep-mutant viruses release virions more readily or more completely under alkaline conditions, than wild-type virus. Scanned images of the sucrose gradient banding patterns were used to quantify the ratios of virions containing different numbers of nucleocapsids, for each virus. We can conclude from the figures that there is no obvious difference between viruses in the numbers of virions containing few versus many nucleocapsids.

ACKNOWLEDGMENTS

The authors thank Dr. George F. Rohrmann, Oregon State University, for providing the OpMNPV PEP polyclonal antibody, Andres J. Lopez at Iowa State University for help on use PAUP* software, Tracey Pepper and John Mattila at the Bessey Microscopy Facility, Iowa State University for assistance with electron microscopy work. This research was supported by Hatch Act and State of Iowa funds. 34 FIG. 1. Construction ofRoPEPLacZ and RoPEPMTG-TAG. (1) The RoMNPV-Rl HindIII-G fragment containing pep gene. (2) The hsp70 promoter-lacZ cassette was inserted into the pep gene to produce RoPEPLacZ. (3) The start codon of the pep gene was replaced and a stop codon introduced 37nt downstream to produce RoPEP!iATG-TAG. Mutations made to the coding sequence to produce RoPEP !iATG-TAG and the predicted amino acid sequences are shown. Two additional viruses RoPEP!iATG and RoPEPTAG were also made with single mutations (not shown). 35

.Xba 1,4974 .Hind 111, 1 ;Bg/ 11,4985 : .Xba 1,961 1 .EcoR V,5961 : : Bgl 11,1700 : : Hind 111,7578 (1) RoMNPV-R1

..... (pep) polyhedral envelope protein

(2) RoPEPLacZ

.Hind 111, 1 .Bgl 11/BamH 1,8725 : .Xba 1,961 .Xba 1,4974 . .EcoR V,9701 : : IBg/ 11,1700 : (indlll,5400 : (ind 111,11318

~0-/a/ pep

TCG--..TAG

.Xba 1,4974 .Hind 111, 1 ;Bgl 11,4985 : .Xba 1,961 ~ .EcoR V,5961 : : Bgl 11,1700 : : Hind 111,7578 (3) RoPEP ~ATG _TAG .______..._...... ______...... , _ ____... _____.

.....pep

wild-type CAA AAT ATG AAA CCG ACG AAT AAC GTT ATG TTC GAC GAC GCG TCG GTC CTT PEP Met Lys Pro Thr Asn Asn Val Met Phe Asp Asp Ala Ser Val Leu CAA AAT ATC AAA CCG ACG AAT AAC GTT ATG TTC GAC GAC GCG TAG GTC CTT RoPEP~ Lys Pro Thr Asn Asn Val Met Phe Asp Asp Ala * Val Leu ATG_TAG *

FIG. 1 36 FIG. 2. Alignment ofbaculovirus polyhedral envelope protein sequences. The protein sequence of RoMNPV PEP was aligned with homo logs from AcMNPV, BmMNPV, CfNPV, EppoMNPV, HaSNPV, LdMNPV, OpMNPV, SeMNPV and SpltMNPV. Dashes (-) indicate gaps; amino acid number is shown on the left; the most commonly used amino acid at each position ("majority") and amino acid number are shown above the alignment. Numbers after the name of each baculovirus represent the ORF number on the complete genome. Shaded residues indicate the most common residues at each portion. 37

------KK- MDPTNNVMFDDASVLWI DADY! YQNLKMPLSTFQQLLF Majority

10 20 30 40 50 ------~K~ i RoMNPVPEP ------~K~j AcMNPV131 ------~K~j BnMNPV108 ------MS S C ~V ~i C1MNPV3 EppoMNPVl 16 HaSNPV120 LdMNPV136 OpMNPV129 SeMNPV46 SpltMNPV132

60 70 80 90 100 39 llA~-----111-----111 RoMNPVPEP 39 Ii.Al------~i~i~- ---- ~J1Hi AcMNPV131 39 llAl------111-----~ll BnMNPV108 43 111 !jjl------TiliPFPPGNl00i C1MNPV3 43 1111----NPPlllFPPNNTI llNll EppoMNPVl 16 46 - LAP KRCWS KHHN- -- HRIR- --- LIDNITF ~ ' ILF HaSNPV120 48 - ~iiiHT LdMNPV136 39 s ~iil~l~l, ~ ~~~~~~~~;~;~~~~~~~~~~~ ···· · I OpMNPV129 44 - ~irniQ K C R- --- CP HHHii- --- ~iDGGIVF I Li SeMNPV46 48 - V~ j P RllRCL G RCS- --- HTIR- --- FDNN~VF SpltMNPV132

SLFADQLLTTFI ANNY------LSYFSRRRSSPSRSR Majority

110 120 130 140 150 77 RoMNPVPEP 77 HI:: : : : : : : : : : : : : : : : : : ~I I ~ AcMNPV131 77 II'------l ,c BnMNPV108 86 H i------M. C1MNPV3 88 Hi:~i~i~i H------c EppoMNPVl 16 88 lllQLllELl~ELAESRRRSQSRSCSRSQSRSR. llvsRNRi HaSNPV120 92 lcl~lv A~------RDACCAQPP---APIPDYNAllPDCI LdMNPV136 88 ~i~l!~i~HlT 11:_------·:~iilc iPc~j QPPC OpMNPV129 84 1 SeMNPV46 88 v~l~~· ~~ I ~ : ~II ~:~=~~~~=~~~=~~: ~~::! ~:. . H·;::~:1.:~ SpltMNPVI32 S ------R- -- RS ---- RP RS RC RS RS R- -- R------QI FD Majority

160 170 180 190 200 108 IRsRS--PHCIPRsllP-Hclli isRslssSPRRG--RR~ 108 lsRslsssPRRG--RR~ AcMNPV131 108 1~~~~~~~~~~~~~11:::~111 si i,sRslssSPRQG--RR~ BnMNPV108 116 ------Q!lilJiF D~jA C1MNPV3 118 P ------vf.!i i orn~ ! A ~ ~ :: : : : : : : ~ :~ :: : : I j,JiJ:M EppoMNPVl 16 138 RSRSRSNSRGIRRsllNsRGIRll~sl ITcHIRR------RTSE HaSNPV120 131 PPL P ------fj!jQP P ~jKP ------NDS E LdMNPV136 119 P ------Qj1\ijp FD~!AQT------iJJiJ!L i OpMNPV129 130 SeMNPV46 136 ~~~:=~=~~~1:~~~~~~=:1::1~1~:!:::::::=~~~~~~~~~~;~~ SpltMNPV132

FIG. 2 38

ALEKIRHQNDLLVNNVNQI SLNQTNQFLELSNMLNAVRAQNVQLLAALET Majoricy

210 220 230 240 250 153 RoMNPVPEP 155 1111 H l l : IH i l~~ 1 AcMNPV131 142 ~ : ~ ~ii i ~ i Hi. : ~M~ i BnMNPV108 129 ~ i.U ii i G~H : i ~i ~ i G~ C1MNPV3 132 I ~LGlllE l lITGI EppoMNPVl 16 179 Y H is RH i ~ ~i ~ i ~ i s A~ HaSNPV120 146 L R ~ !!i v R fj ~ i 1 L IG L LdMNPV136 133 .. iLARls v~ i ~SL OpMNPV129 171 L ll R I TR~ · ILTSI SeMNPV46 186 QN I A I SK~ ETI Liii. SpltMNPV132

AKDAILTRLNTLLDEITDLLPDLTSQLDELAEQLLDAINTVQQTLRNELN Majoricy

260 270 280 290 300 203 s ~ i ~ i! ~~ ! H J! M ~i lK RoMNPVPEP 205 s H : i~ i H i! M ~i ~K AcMNPV131 192 s H i ~~i~L M~! IK BnMNPV108 179 ~m A ~i ~ i: ~~ i H l; C1MNPV3 182 ~ jjj K~AH J IQs E ~ Q . D ~ EppoMNPVl 16 229 LLESVDRQllll'PLJ.t RLNL ~j SSEVRQI NQFSG~ ! lw. i AES RFID HaSNPV120 196 I F~DGV- - ~ l SG~ j DEKLSR~JI A~~ j DGHFADFGSAjjjl j DA~LAQLID LdMNPV136 183 TIH~ i!i ~ i ~iH ! IA~ ! vlo~i il KAAH ! IQs Al~ ! Q~!~ i ~oKll~i H!~ iii ~s ~ i AI OpMNPV129 221 ILETVEG- IGDITGDF I RllAEIDTRIAAl~TTI I NlllQLSDQ SeMNPV46 236 IQEl l EIGFINVEQSLESIIAGIETRISSALNAIN~ILVRLIE IV SpltMNPV132

NTNSILTNLASSITNJNGTLNNLLAAIENLNGGGGGG------LNEADR Maj~cy

320 330 340 350

253 RoMNPVPEP 255 AcMNPV131 242 BnMNPV108 229 C1MNPV3 232 EppoMNPVl 16 279 HaSNPV120 244 LdMNPV136 233 OpMNPV129 270 SeMNPV46 286 SpltMNPV132

QKL DL VL TL VT EI KN! LT GT LT------Majority

360 370 RoMNPVPEP 298 y 1 lll i ~N ~ : ~ 111 1~ 1m 299 Y~lill \ ~N ~ J l JiJlllJil AcMNPV131 292 BnMNPV108 268 C1MNPV3 275 EppoMNPVl 16 322 D~~~[: i~~~i!~~~~~~R ~R K. HaSNPV120 287 STVIRI G PEI VAAAAAAAAKRAH . LdMNPV136 275 IAI NE l l RlllMllARK. OpMNPV129 310 ~ : TI NT I D L QT ~ il i L G~ ii !l \ QP NI PL NKK. SeMNPV46 SpltMNPV132 329 ll1IA1IE1L I PPLRK.

FIG. 2 (continued) 39 FIG. 3. Unrooted phylogram derived from analysis ofbaculovirus PEP proteins. Amino acid sequences were aligned with ClustalW method. The phylogram tree was created with neighbor joining methods by PAUP* version 4.0b 10. Baculoviruses are divided into two groups. One group (group I) includes RoMNPV, AcMNPV, BmMNPV, CfMNPV, EppoMNPV and OpMNPV; the other group (group II) includes HaSNPV, SpltMNPV, LdMNPV and SeMNPV. Group one is more conserved than group IL Bars indicate 0.05 changes (5% divergence) or 0.1 changes (10% divergence). AcMNPV and RoMNPV are the most closely related baculoviruses of those included in the analysis. The ORF number ofpep in each baculovirus genome is shown. The AcMNPV, RoMNPV and BmMNPV branches are enlarged to show the tiny branch better. 40

---- 0;05 changes_

EppoMNPVl 16

CtMNPV3 OpMNPV129

AcMNPV131 RoMNPV124 _ BmMNPVl08 HaSNPV120

SeMNPV46

SpltMNPV132

LdMNPV136

---- 0.1 changes FIG. 3 41 FIG.4. Analysis ofrecombinant virus genomes by restriction enzyme digestion. The genomes of the pep-mutant viruses and RoMNPV Rl were restricted with HindII.I, and electrophoresed on a 0.8% agarose gel. A 1 kb DNA ladder was loaded as standard. Fragment sizes are shown on the left. RoPEPLacZ has additional 5.9 kb and 5.4 kb (shown by arrow heads) HindIII fragments that are absent from the other virus genomes, and lacks the 7.6 kb fragment (shown by arrow) that is present in the other virus genomes. 42

FIG. 4 43 FIG.SA. Analysis of virus proteins in cultured cells by SDS-PAGE. Sf9 cells were infected

with AcMNPV-C6, RoMNPV-Rl, RoPEP~ATG, RoPEPTAG, RoPEP~ATG-TAG and RoPEPLacZ. Cells were harvested at 72 hours post-infection. Each lane was loaded with about 5x 1o4 cells. Uninfected Sf9 cells were run as control for identification of virus proteins. Molecular mass standards are shown on the left and the position of polyhedrin is indicated. SB. Western blot analysis of PEP expression in cultured cells. Sf9 cells were infected

withAcMNPV-C6, RoMNPV-Rl, andpep-mutantRoMNPVs (RoPEP~ATG,

RoPEPTAG, RoPEP~ATG-TAG and RoPEPLacZ). Proteins separated by SDS- PAGE were transferred to Hybond-P PVDF membrane, and incubated with OpMNPV-PEP polyclonal antibody. Molecular mass standards are shown on the left. The position of PEP is indicated (arrow). 44

kDa 216- 129- 91-

43.2-

33- ~Polyhedrin

18.8-

7.7-

FIG. 5A 45

(far kDa ~c

91- -

33-

18.8-

FIG. SB 46 FIG.6. Scanning electron micro graphs of polyhedra. Polyhedra were isolated from dead fifth instar H. virescens larvae infected with wild type RoMNPV-Rl or one of the pep- mutant viruses. The micrographs show the smooth surface ofRoMNPV polyhedra, and the pitted surface of the pep-mutant virus polyhedra. (A) RoMNPV Rl, (B)

RoPEP~ATG, (C) RoPEPTAG, (D) RoPEP~ATG-TAG, (E) RoPEPLacZ. Bars, l.Oµm. 47

FIG. 6 48 FIG. 7. Transmission electron micro graphs of polyhedra. Sf9 cells were infected with (A)

RoMNPV-Rl, (B) RoPEP~ATG, (C) RoPEPTAG, (D) RoPEP~ATG-TAG and (E) RoPEPLacZ. Cells were harvested at 72 hours post infection The electron dense polyhedral envelope is only apparent in (A). Bars, 200 nm. 49

FIG. 7 50 FIG. 8. Immunogold labeling of PEP. Electron micrographs of polyhedra in Sf9 cells infected with (A) RoMNPV Rl, (B) RoPEPMTG, (C) RoPEPTAG, (D) RoPEPMTG-TAG and (E) RoPEPLacZ. PEP labeled with lOnm gold particles is localized on the surface of polyhedra in A and B only. Details of surface labeling are shown. Bars, 200 nm. 51

Fig. 8 52

FIG. 8 (continued) 53 FIG. 9. Sensitivity of polyhedra to alkali. Insect derived polyhedra of RoMNPV Rl and pep- mutant viruses were incubated in 0.05M Na2C03 for 2, 5 or 10 min. (A) RoMNPV-

. Rl; (B) RoPEP~ATG; (C) RoPEPTAG; (D) RoPEP~ATG-TAG; (E) RoPEPLacZ. Virions were released from pep-mutant viruses within 2 min, while RoMNPV Rl polyhedra retained their integrity without virion release following a period of 10 min in alkali. Bars, 200 nm. 54

Omin ..,r·.·. ~· · ··..

c

FIG. 9 55

Omin 2min lOmin

D ,

E

FIG. 9 (continued) 56 . FIG.10. Ostrinia nubilalis and Trichoplusia ni midgut pH analysis with broad range pH indicators. Rows A and B, show the color change of meta-cresol purple and indigo carmine respectively at different pH; row C, natural color of laboratory-reared larvae; row D, shows the gut coloration following ingestion of 0.05% meta-cresol purple; row E, shows the gut coloration following ingestion of 0.5% indigo carmine; rows F and G, show the color change of acid fushin and aniline blue respectively at different pH; row H, shows the gut coloration following ingestion of 0.1 % acid fushin; row I, shows the gut coloration following ingestion of 0.2% aniline blue. These results indicate that the gut pH of these two species is within the range of 9 - 1 I. 57

PH 7.0 8.0 9.0 11.5 13.0

A

B

Ostrinia nubilalis Trichoplusia ni

c

D

E

FIG. 10 58

10.0 11.0 12.0 13.0 14.0

G

Ostrinia nubilalis Trichoplusia ni

H

I

FIG. 10 (continued) 59 FIG.11. Comparison of the number ofvirions encapsulated within polyhedra. The numbers of virions per polyhedral cross-section were quantified for each virus. The results are presented as the average number of virions per µm2 of polyhedral cross-sectional area. Data presented represent the mean± standard deviation of 30-41 cross-sections examined for each virus. There are significantly more virions in RoMNPV Rl compared to the pep-mutant viruses. Each bar labeled by the different letter is significantly different with each other at p<0.05 based on Tukey's and Bonferroni tests. 60

18.00 a 16.00

14.00

Cll c: 12.00 0 'C: 10.00 .....> 0 I.. 8.00 .cCl> E :::s 6.00 z 4.00

2.00

0.00 RoMNPV-R1 RoPEP.6ATG RoPEP TAG RoPEP MTG- RoPEPLacZ TAG Polyhedra

FIG. 11 61 FIG.12. Preliminary examination of the numbers ofnucleocapsids in occlusion-derived virions for pep-mutant and wild-type viruses. Occlusion-derived virions in 25-60% continuous sucrose gradient are shown. White bands were formed by purified virions containing the same number of nucleocapsids. The highest band (band 1) is assumed to contain virions with one nucleocapsid. The lowest band is assumed to contain the largest number (-9) of nucleocapsids per virion. Graphical analyses of the intensities of the virion bands in each tube are also shown. Graphs were created by Scion Image based on band intensity. The area of each peak under the curve reflects the proportion of ODY in each band. 62

FIG. 12 63

Table 2 Lethal concentration data for wild-type and pep-mutant RoMNPV in first instar Ostrinia nubi/alis and Trichoplusia nt

Host Virus LCso a (95% Cl) Slope Heterogeneity in OBs/µl (x2/D.F.)

0. nubilalis RoMNPV 6611 a (1,631-17,445) 1.03 1.29

RoPEPMTG 37,934 b (25,629-55,599) 1.88 0.32

RoPEPTAG 21,582 b (14,212-32,516) 1.49 0.39

RoPEP MTG-TAG 27,898 b (9,483-85,017) 1.21 1.66

T. ni RoMNPV 88 a (59-131) 1.51 0.24

RoPEPMTG 122 a (85-174) 1.95 0.25

RoPEPTAG 138 a (49-392) 1.88 2.12

RoPEP MTG-TAG 90 a (46-180) 1.78 1.12

aData analyzed by probit analysis (Russell et al., 1977). Probit-dose relationship is linear and data fit probit model by x2 test at P = 0.05 confidence level (Robertson & Preisler, 1992). bLC50 estimates for the same host followed by different letters are significantly different at P = 0.05 confidence level by dose ratio comparison test (Robertson & Preisler, 1992). Data are representative of two other replicates. 64

Table 3 Ratios of band intensities for ODV containing different numbers of nucleocapsids. The areas for each peak in Fig. 12 were calculated using Scion Image and the area of the first (top) peak taken as 1.

Peak RoPEPLacZ RoPEP!iATG RoPEPTAG RoPEP tiATG-TAG RoMNPV-Rl 1 1 1 1 1 1 2 1.65 1.17 1.10 1.24 1.67 3 4.07 3.25 3.16 3.51 3.85 4 7.55 5.11 4.62 5.18 7.61 5 7.51 5.13 4.64 5.27 9.46 6 6.39 4.42 3.87 4.38 8.97 7 4.32 2.91 3.57 7.10 8 2.15 4.96 65

CHAPTER 3. GENERAL DISCUSSION

Baculoviruses have a narrow host range, and most only infect one species or genus. AcMNPV, the most studied baculovirus, is one of a few baculoviruses that have a relatively broad host range. AcMNPV can infect more than 30 insect species (Cunningham, 1995). RoMNPV (also known as AfMNPV) is closely related to AcMNPV and also has a broad host range (Harrison & Bonning, 1999; Hostetter & Puttler, 1991; Lewis & Johnson, 1982). RoMNPV is the most effective virus for 0. nubilalis control in terms of lethal time, lethal concentration, and has great potential for management of numerous other agriculturally important pests (Hostetter & Puttler, 1991).

Mutation of AcMNPV pep was reported to reduce the LC50 of the virus six-fold compared to AcMNPV in first instar T.ni (Ignoffo et al., 1995). Mutation of the pep gene in LdMNPV however had no significant effect on virulence (Bischoff & Slavicek, 1999). RoMNPV shares high identity with the AcMNPV genome (Harrison & Bonning, 1999), and RoMNPV PEP has 99.4% identity with AcMNPV PEP. Hence it was expected that we would obtain a similar result on mutation ofRoMNPV pep as was seen for mutation of AcMNPV pep. Based on our results however, removal of PEP from RoMNPV polyhedra is clearly not a good strategy for optimization of virulence. The absence of PEP may result in loss ofvirions from the periphery of polyhedra, thereby reducing virulence against semi-permissive species. In addition, or as an alternative hypothesis, variation in degradative midgut factors between the permissive and semi-permissive species, may contribute to the loss of virulence ofpep- mutant viruses in the semi-permissive species.

Genetic engineering is an attractive approach for enhancing baculovirus insecticides for commercialization. American Cyanamid and DuPont both pursued development of recombinant baculoviruses as biological control agents (Black et al., 1997). Although these commercial programs were terminated, the recombinant baculoviruses developed were competitive with Bt and some chemical insecticides under field conditions, and had a projected market share of $40 million per year. Although such a profit margin is inadequate 66

for a product of a large multinational agricultural industry, smaller start-up companies may exploit recombinant baculovirus products in years to come. Recent progress in baculovirus genomics and proteomics will facilitate our understanding of gene regulation and function, which will enhance the potential for genetic optimization in the future.

Determination of the functions of all baculovirus genes will facilitate future engineering ofbaculoviruses for enhanced virulence. Our current knowledge of genes involved in virulence is inadequate. Genes involved in determining baculovirus host range that have been characterized to date have no practical application for enhancing baculoviruses as insecticidal agents (Miller & Lu, 1997).

However, with the sequencing of the genomes of related baculoviruses, it will become possible to identify genes that have undergone adaptive evolution that may account for differential infectivity to different species ofLepidoptera. For example, RoMNPV and AcMNPV are very closely related, but RoMNPV is significantly more virulent against several agricultural pests including the corn earworm Helicoverpa zea, the European corn borer Ostrinia nubilalis, and the navel orangeworm Amylois transitella, when compared to AcMNPV (Hostetter & Puttler, 1991; Lewis & Johnson, 1982). Analysis of the 63 genes that are common to the baculovirus genomes that have been sequenced revealed two genes that have undergone adaptive evolution (Harrison, unpublished). The function of rol 02/acl 07 is unknown, but rol 35/acl 43 encodes a protein found on the envelope of occluded virus. Production of recombinant AcMNPV with ro102 or ro135 followed by bioassay in species showing differential susceptibility to the two viruses could be used to determine whether these genes have a role in virulence.

In addition to genetic manipulation ofbaculoviruses for increased virulence, it is also likely that additional chitin binding reagents will be identified that could be used in baculovirus formulations. These reagents would act in the same way as the fluorescent brightener M2R, to block formation of the peritrophic matrix barrier to increase the efficiency of primary infection of midgut cells. Indeed, chitin-binding peptides may also have 67 potential for use in development of insect resistant transgenic plants because disruption of the peritrophic matrix inhibits feeding. 68

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ACKNOWLEDGEMENTS

I would like to give my special thanks to my major professor, Dr. Bryony C. Bonning, for her guidance, advice, and encouragement during the last three years. I also thank my POS committee members, Dr. Susan L. Carpenter and Dr. Jeffrey K. Beetham, and also Dr. W. Allen Miller and Dr. Russell Jurenka, for their comments and help during my graduate career. I thank Dr. Robert L. Harrison, and Dr. Anthony J. Boughton for their assistance with lab work and many brilliant ideas on my research project. Finally, I am grateful to my husband, Pengcheng Lu, my son, Jerry Lu, my father, Y aozu Jin, and my mother, Daifen Yu, for their love and support.