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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John's Road, Tyler’s G reen High Wycombe, Bucks, England HP10 8HR 77-10,522 ELLIS, Beth-Jayne, 1942- INFECTIVITY OF A NUCLEAR POLYHEDROSIS VIRUS IN GALLERIA MELLONELLA fL .l LARVAE, WITH EMPHASIS ON STABILITY AND RELATIVE INFECTIVITY OF NON­ OCCLUDED VIRUS PARTICLES. The Ohio State University, Ph.D., 1976 Microbiology

Xerox University Microfilms,Ann Arbor, Michigan 48106 INFECTIVITY OF A NUCLEAR POLYHEDROSIS VIRUS IN GALLERIA MELLONELLA (L.)

LARVAE, WITH EMPHASIS ON STABILITY AND RELATIVE INFECTIVITY OF NON-OCCLUDED VIRUS PARTICLES

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Beth-Jayne E llis , B.A., M.Sc.

The Ohio State University

1976

Reading Committee: Approved by Donald H. Dean W illard C. Myser Advisor Gordon R. S tairs Department of Entomology ACKNOWLEDGEMENTS

I am grateful to Prof. G. R. Stairs, whose guidance and support directs students to evaluate their own work and be observant in life. I thank my exam and reading committees, Drs. Donald H. Dean, James I . Frea,

Willard C. Myser, Walter C. Rotheribuhler, and Gordon R. Stairs, for their suggestions and critical evaluation of this dissertation. Recog­ nition goes to Dr. W. B. Parrish for taking the electron micrographs.

I wish to express my gratitude to Professors James H. Oliver and Edwin T. Hibbs at Georgia Southern College for their encouragement and cooperation while I completed this work. Support and financial assist­ ance for this study were provided, in part, by my husband, and my father, Frederick D. Obenchain, and Gilman C. E llis , respectively. VITA

1942...... Bom: Portland, Maine 1965 ...... B.A., University of Maine, Orono, Maine 1965-1968 ...... Medical Technologist (ASCP) : Animal Research Lab., University Hospital, and Children’s H ospital, Columbus, Ohio 1967 ...... M.Sc., The Ohio State U niversity, Columbus, Ohio 1968-1970 ...... Research Assistant, Invertebrate Pathology Lab., Dept. Entomology, The Ohio State U niversity, Columbus, Ohio 1970-1973 ...... Instructor/Lecturer and Teaching Associate, General Biology Program, The Ohio State U niversity, Columbus, Ohio

1973, 1975-1976 ...... Visiting Consultant, Biology Dept., Georgia Southern College, Statesboro, Goergia

i i i CONTENTS Page

ACKNOWLEDGEMENTS...... i i

VITA...... i i i INTRODUCTION...... 1 TERMINOLOGY, CLASSIFICATION, AND NOMENCLATURE...... 3 LITERATURE REVIEW...... 7 MATERIALS AND METHODS...... 26 V irus...... 26 The maintenance of sGalleria iiiellbriella (L.) ...... 26 Enzymes...... 27 Alkaline solution...... 27 Centrifugation ...... 28 M icrofiltration ...... 28 Microinj ect ion ...... 28 Light microscopy...... 28 Electron microscopy...... 29 Sucrose gradients ...... 29 Titration of virus ...... 29 Cell counts ...... 29 Expressions of virus concentration ...... 30 RESULTS...... 31 Effect of storage and serial passage...... 31 Storage...... 31 Prolonged alkaline treatment ...... 32 Incubation with sodium chloride ...... 33 S eria l Passage...... 33

Virus and polyhedral replication and development in hemocytes.. 34 Infectious virus in hemolymph of diseases larvae ...... 35 Effect of filter pore-size, dilution, and serial passage.. 35 Electron microscopy observations ...... 37 Effect of DNase on infectious activity ...... 37 Effect of RNase on infectious activity ...... 39 E ffect of alkaline treatment on in fe c tiv ity of hemolymph.. 40

iv DISCUSSION 41 Storage and serial passage ...... 41 F iltra tio n ...... 43 Electron microscopy observations ...... 45 Virus multiplication in early disease ...... 46 Possible role of nucleic acids....' ...... 48 Interference of host development ...... 50

CONCLUSIONS...... 53 SUM4AFY...... 56 TABLES...... 57

FIGURES...... 76 APPENDIX A...... 83 APPENDIX B...... 87 LITERATURE CITED...... 89

v 1

INTRODUCTION

The nuclear polyhedrosis viruses (NPV) are agents of certain viroses in Lepidoptera, Hymenoptera, D iptera, and Orthoptera (Harrap, 1973; Ignoffo, 1973). A distinctive cytopathological characteristic of this disease is the development of polyhedral virus inclusion bodies in the nuclei of susceptible host c e lls (Harrap, 1972a, b, c ) . Most knowledge of NPV is from work on Bombyx mori NPV due to the importance of this in­ sect to the silkworm industry in Japan, France, and Italy. More recently, attention has been given to the NPVs of cabbage loopers, com borers, and other crop-destructive insects (mostly Noctuidae). These viruses show promise as agents for the control of host insects. Although poly­ hedral viruses, nuclear and cytoplasmic, are the most common causes of in sect v iro ses, knowledge o f these insect viruses is lim ited compared to knowledge o f vertebrate, p lan t, and bacteria viruses.

The enveloped, rod-shaped particles of NPV survive outside the host when occluded in a crystalline protein matrix (polyhedral). This form of the virus is eaten by larvae and is responsible for the zootiological spread of the disease. Virus multiplication and spread from cell to cell within a larva appears to be caused by non-occluded virus particles.

Virions freed from the occlusion matrix under alkaline conditions may also induce viral disease when injected or fed to larvae. The spread of infection within a larva may be due to the presence of non-enveloped virus within a larva (Kawarabata, 1974). Sucrose gradient techniques indicate that the smallest infectious NPV particle from Galleria mellonella hemolymph is less than 50 nm in diameter (Dimmitt, 1968). In cell cultures, non-occluded virions are more susceptible than alkaline-released occluded particles as a source of infectivity (Vaughn,

1968). Vaughn (1965) also presented chromatographic evidence for differ­ ences between particles which had been chemically released from their occlusion matrix and non-occluded virus particles obtained from insect hemolymph. If these naturally free virions are responsible for the intracellular transmission of nuclear polyhedrosis disease, then some knowledge of their stability and infectivity is fundamental to the under­ standing of this disease. Occluded particles of NPV over-winter in the field to produce new infections in the next year, but levels of activity decreased over long periods of time (Jaques, 1967; 1974). Alkaline-released virus is not suitable for storage (Harrap and Longworth, 1974), but there is no report on storage of non-occluded infectious virus particles of G. mellonella.

This dissertation is concerned with the stability of variolas forms of the NPV of the greater wax moth, G. mellonella, and the relative infectivity of these forms. Also, patterns and rates of disease develop­ ment at the cellular level were investigated. 3

TERMINOLOGY, CLASSIFICATION, AND NOMENCLATURE OF INSECT VIRUSES

Definitions of viruses.--Current views on the nature of viruses re­

turn to early broad definitions (Wildy, 1971). With the first passage of plant (Iwanowsky and Beijerinck, studying tobacco mosaic disease) and animal viruses (Loeffler and Frosch, studying cattle foot-and-mouth disease) the agent of any non-cellular infection that could be trans­ ferred to a new host to produce a new infection was considered to be viral in nature, or a "contagium vivum fluidum". Toxins were also in­ cluded in this concept. A more recent and general working definition of

a virus is quoted as follows:

Particles made up of one or several molecules of DNA or RNA, and usually but not necessarily covered by protein, which are able to transmit their nucleic acid from one host cell to another and to use the host’s enzyme apparatus to achieve their intracellular repli­ cation be superimposing their information on that of the host cell; or occasionally to integrate their genome in reversible manner in th at o f the host and thereby to become cryptic or to transfer the charac­ teristics of the host cell. (Fraenkel-Conrat, 1969)

Variations of this definition emphasize various aspects of virus

inabilities as well as specialities, e.g., host-cell dependence, one type of nucleic acid (DNA or RNA), lack of a Lipmann system (cellular organi­ zation and enzyme system) (Lwoff, 1957), and self-direction (Luria, 1959).

Agents such as rickettsia, psittacosis-like particles, mycoplasms, and chlamydia are not considered to be viruses due to the presence of two •types of nucleic acid (DNA and RNA), a partial wall, or enzyme systems. Luria and Darnell (1968) used a definition which emphasized the importance of virion: "contain the viral genome and transfer it to other cells". It is now known that transfer factors and viriods [e.g., potato spindle tuber virus (Diener, 1965; Diener and Raymer, 1969)] consist of naked nucleic acid and do not form virions. The existence of such particles stresses the need of returning to broad definitions. Terminology.--Terms used to identify the various structural compo­ nents of a virus throughout this paper follow the suggestions of several workers (Lwoff e t a l . , 1962; Caspar e t a l ., 1962). The nucleic acid and its surrounding capsid are collectively referred to as a nucleocapsid, which may be either naked or enveloped. Based on EM studies, the outer membrane (developmental membrane of Bergold, 1963b) of NPV and GV is called an envelope and the inner membrane (intimate membrane of Bergold) is con­ sidered to be a part of the capsid (Summers and Amott, 1969; Stoltz et^ a l., 1973; Harrap, 1972b). NPV "strains" may be singly-embedded or multiply-embedded, that is, there may be one or several nucleocapsids within a single envelope. Some investigators consider each bundle (all those rods within an envelope) to be a single virion (Kawanishi and

Paschke, 1970), whereas others call each rod (nucleocapsid) a virion or a particle. Just as there is some question as to whether a single nucleocapsid of a multiply-embedded NPV should be called a virion, there is a question as to whether the inclusion protein should be considered as an integral part of the complete or mature virus. The restrictive definition of a virion as a "complete virus particle" presents a similar problem when applied to other occluded viruses, e.g., adeno-virus type 5, and many insect viruses. Because of these difficulties, the terms virus form or virus particle will be used in preference to the term virion in these studies. C lassificatio n and Nomenclature. - -Although there is not enough in ­ formation to classify viruses according to their phylogenetic relation­ ships, a system of reference is needed to minimize confusion. The International Committee on the Taxonomy o f Viruses (ICTV), formerly the Committee on Nomenclature of Viruses (ICNV) has accepted a Unitarian approach of grouping viruses according to the nucleic acid of the genome (DNA or RNA), the molecular weight of the virus particle, and other mor­ phological characteristics (Fenner, 1976; Wildy, 1971). This is an extension of an earlier scheme by Fenner (1968) based on the attributes of the dormant virus, the virion. In contrast are dynamic features (Subah-Sharpe et a l., 1966) . Opposition to a Unitarian approach which includes the insect viruses among all other viruses is based on the uniqueness of many of the insect viruses, namely the crystalline lattice structure of their inclusion bodies and nucleic acid composition of the viral genomes (see Literature Review Section). In the currently accepted classification scheme (ICTV), as in the earlier and more restringent sys­ tem of Lwoff, Home and Toumier (IHT) (1962), insect viruses are distri­ buted widely among the groups of RMi and DNA viruses of plants, animals and bacteria (Table 1, 2). Within these groups, occluded insect viruses are further subdivided on the basis of the morphology of th e ir inclusion bodies, i.e ., Baculovirus A and B. Another classification and nomenclature system has been proposed by

Harrap and Tinsley (1971). Their distribution of insect inclusion body viruses is an extension of the LHT System: type of nuclei acid (DNA or RNA) for subphylum, capsid symmetry (helical or icosahedral) for class, presence or absence of envelope (enveloped or naked) for order. Family names of insect occluded viruses in DHE, order Chitovirales, are Nupoviridae, Granoviridae, and Arthropoxviridae for NPV, GV, and entomo- pox respectively. The generic name of NPV of the was moth, Galleria mellonella, in this system is Nupovirus mellonella (Table 1 and 3). In the international system, the same virus is Baculovirus sp., in the family Vaculoviridae. 7

LITERATURE REVIEW

The nuclear polyhedrosis virus (NPV) of Galleria mellonella is a Baculovirus (Fenner, 1976). As a genus of family Baculoviridae, the NPV baculoviruses have distinctive characteristics, yet they have similarities to other Baculoviridae as well as other virus groups, DNA and RNA viruses, including phages. Nucleic Acid Content Nuclear polyhedrosis virus (NPV) and granulosis virus (GV), the two subgroups of Baculoviridae, contain covalently, closed, supercoiled, double-stranded DNA with a molecular weight of approximately 80 x 10^ daltons (Summers and Anderson, 1972a, b; 1973). In contrast, the double­ stranded circular ENA of iridescent virus has a reported molecular weight of 130 x 106 daltons (Kelly and Robertson, 1973). Several other circular genomes are known to exist in viral and non-viral DNA's (Helinski and Clewell, 1971). The molecular weight of the DNA insect viruses varies from 1.2 to 160 x 10^ daltons (Table 4), the same range as for all DNA viruses (Wildy, 1971). The ratios of A/T and G/C in baculoviruses from different insects are approximately 1.0 in all cases studied, but the ratio of adenine + thymine to guanine + cytosine varies (Wyatt, 1952b). Groups having sim­ ilar AT:GC ratios are from strains of hosts belonging within a single family. This is unlike the DNA of animals and strains of plant viruses in which there is a distinctive composition for each species and strain, respectively (Smith and Markham, 1950; Wyatt, 1952b). Two baculoviruses with different AT:GC ratios and different morphology infecting the same host suggests a degree of host and virus nucleic acid independence which is seen in certain phages (Smith and Wyatt, 1951). There are conflicting reports regarding the presence of RNA in NPV

(Kreig, 1956; Smith and Wyatt, 1951; Tarrassevich, 1946; Wyatt, 1952a, b;

Yagi e t a l . , 1951. The nucleotide bases reported from Bombyx mori NPV differed from host RNA (Table 5) (Eto, 1955; Faulkner, 1962). Uracil has been identified in the DNA genome of herpesvirus (Hirsch and Vonka, 1974) and the presence of RNA in the genetic material of bacteriophages

T4 and T5 has been demonstrated (Rosencranz, 1973; Speyer et a l., 1972). A possible relationship between the presence of RNA and the fragmentation of DNA under alkaline conditions has been suggested for both the phages and herpesviruses (Davis et al., 1972). Inactivation of polio virus and tobacco yellow mosaic virus is associated with breakage of RNA in the capsid under alkaline conditions (Kaper and Halperin, 1965; Boeye and Elsen, 1967). The location of RNA associated with the NP virus is unknown.

Morphology and Structure The NPV nucleocapsid is rod-shaped (20-70nm x 200-400nm). The granulosis viruses also have rod-shaped particles and may have a DNA central core as described for the virus rods of NPV (Amott and Smith, 1968). The other group of rod-shaped double-stranded DNA viruses is the poxviruses (160 x 10^ daltons). Rod-shaped DNA phages (fd, fl, M13) are 1.7 x 106 daltons and single-stranded. The rod form is also found among RNA v iru ses, including some plant viruses (Home et^ a l ., 1959; Klug and

Caspar, 1960; Vela and Lee, 1974), rabies, sigma -virus of Drosophila, Vescicular Stomatitis Virus of cattle, RNA. tumor viruses, influenza,

measles, mumps, Newcastle disease, and canine distemper (Home et al.,

1960). The other insect polyhedral virus, cytoplasmic polyhedrosis virus (CPV), is an icosahedral, RNA virus (Reoviridae) and has 12 projec­

tions on the surface of its capsid, in a pattern similar to that of

adenoviruses (Amott et a l., 1968). The nucleocapsid of NPV is higher in arginine, glycine, alanine, serine, and threonine than polyhedron protein; the two proteins are similar in amount of aspartic acid, pro- line, cystine, and methionine (Wellington, 1954). A protrusion, lOu in diameter and apparently attached to the inner protein layer (intimate membrane), has been observed at one end of NPV

and GV rods (Bergold, 1963a; Bird, 1957, 1959; Kreig, 1963; Ponsen et a l., 1965; Smith, 1962; Summers and Pas dike, 1970). Virus rods of an NPV infecting the sawfly Diprion hercynia were observed in host nuclei with

these processes oriented toward the chromatin material, a behavior which resembles that of bacteriophages during the infectious process. It is not known, however, whether these processes are related to the architec­

ture of the DNA core or whether they are artifacts (Summers and Pas dike,

1970). The only other report of such structures comes from studies of a myxovirus (RHE) observed in tissue culture (Georges and Guedenet, 1974).. With the exception of 1MV and single-stranded DNA phages, rod-shaped viruses, including Baculoviruses, are enveloped (Wildy, 1971). Other enveloped viruses are the togaviruses (RNA encephaloviruses) and the

herpesviruses (DNA) . The envelope of Galleria NPV surrounds bundles of 6 nucleocapsids (Bird, 1964). The envelope structure of most viruses

appears to be formed by budding from cellular membrane structures (Dales, 10 1973). Possible exceptions to this form of development occur in the pox­ viruses Dales and Mosbach, 1968; Granados and Roberts, 1970) and in NPV where the envelope may arise by de novo synthesis in the nucleus (Harrap, 1972c; Morgante et al_., 1974; Stoltz et al_., 1973). A membrane is acquired by individual particles in spaces within the meshwork of the virogenic stroma (Xeros, 1955). There are, however, three reports of NPV budding from the inner layer of the nuclear membrane envelope of infected cells (Bellett, 1968; Bellett and Mercer, 1964; Nappi and Hammill, 1975). En­ velopes of the granulosis virus may be similar to those of NPV (Bellet, 1968) but acquire virus envelopes at the plasma membrane (Robertson et a l., 1974). The envelope of occluded insect viruses may act as a receptor site for the polymerization of the polyhedral proteins (Harrap, 1972a; Harrap and Robertson, 1968; Summers and Amott, 1969), however, CPV is occluded but not enveloped. The NPV envelope of occluded virus may differ from the outer covering of non-occluded nucleocapsids (Harrap,

1970; Summers, 1971). Virus infectivity titrations suggest that the developmental membrane is essential in preserving the infectivity of the NPV virus particle (Bergold, 1963b; Harrap, 1970). An envelope may be necessary for virus attachment to gut cells, but not for the invasion of the Lepidoptera larvae (Khosaka and Himeno, 1972) using hemocoelic inoculations, con­ trary to the conclusion reached earlier (Harrap, 1970). The naturally occuring naked rod from infected hemolymph may be more infectious than a bundle w ith i t s developmental membrane (Kawarabata and Matsumoto, 1973) . In NPV an inner-most layer is 'intimate1 to each virus rod, whereas the outer layer encloses one or several rods within it, depending upon 11 the strain of virus (Bergold, 1953). The outer membrane (envelope) of NPV is possibly a unit membrane (Ponsen et_ a l., 1965; Smith, 1973) where­ as the inner membrane is not a unit structure and appears to be an inte­ gral part of the capsid (Harrap, 1972b). A layer internal to the envelope has been described in several viruses, e.g., GV, iridescent viruses of insects, influenza, parainfluenza, herpesviruses, myxoviruses, and phage PM2 (Bachi et a l ., 1969; Compans and Dimmock, 1969; Hall and Fish, 1974;

Kelly and Robertson, 1973; Kelly and Vance, 1973). In none of these is th is inner lay er a conventional "unit membrane". A ll but NPV are p a r ti­ ally or completely assembled in the cytoplasm of infected cells (Huger,

1963; Kelly and Tinsley, 1973). Current studies of insect inclusion bodies emphasize the structure and formation of these matrices (Bird, 1974; Federici and Anthony, 1972; Summers and Amott, 1969). There is a question as to whether the inclu­ sion body protein is a stru c tu ra l component of the virus p a rtic le o r whether it is simply a host response. No serological relationship be­ tween polyhedra and healthy hemolymph (Krywienczyk and Bergold, 1961).

The formation of the NPV inclusion bodies differs from other insect in­ clusions by the presence of a fibrillar matrix in the nucleus in which the c ry sta llin e la ttic e forms, excluding a l l c e llu la r components (Summers and Amott, 1969). GV appears to lack a fibrillar matrix; CPV has an accretion process of crystal development (Amott et al., 1968) . GV bodies develop by the addition of crystalline protein deposits around the sides until a complete capsule is formed (Amott and Smith, 1968). Analysis of elements showed sim ilarities between the parasporal crystals of Bacillus thuringiensis and the polyhedral inclusion bodies of NP (Faust et a l., 12 1973). The molecular structure of some insect virus inclusion bodies has been described (Bergold, 1963a. The process of incorporation of insect virus particles into polyhedra is highly specific in that the inclusion bodies of NPV do not occlude any cellular components, only virus particles (Stairs, 1968). This specificity does not occur in other inclusion bodies,

e.g., in the chloroplast of a diatom (Holdsworth, 1968). The NPV inclusion bodies tend to be the largest of the insect inclu­ sions, ranging from 0.5u to 15u (Stairs, 1968). Cytoplasmic polyhedra

get as large as 5u in diameter and granulosis capsules range from 0.15u

to 0.5u (Amott et al., 1968; Amott and Smith, 1968; Huger, 1963). NPV shapes vary from dodecahedra, tetrahedra, cubes, and irregular forms and are believed to be specific for a virus species (Amott and Smith,

1968; S ta irs , 1964b). The shape o f the inclusion bodies of insect viruses and T-even phages may be controlled by the viral genome (Aizawa,

1955, Aruga e t a l . , 1961; Bergold, 1963b; Bird and Whalen, 1954;

Gershenson, 1959; S tairs, 1964b, 1968; Yanageda e t a l . , 1970). Shapes of CPV and GV have been separated and maintained in subsequent in vivo in ­

fections (Amott et al., 1968; Stairs, 1964b). CPV inclusion size seems to be related to time (Aruga et a l., 1961) . Polymorphic inclusions of a baculovirus in mosquitos are currently being studied to determine the

control factors of differences in inclusion morphologies (Hall and Fish, 1974). Polyhedral forms of iridescent viruses have been reported

(Williams and Smith, 1958). Crystalline inclusion bodies may be unique to insect viruses (Morgan et al., 1955, 1956; Day et al., 1958). Matrices of vertebrate

poxviruses are noncrystalline (Stoltz and Summers, 1972). A pseudo-lat­ 13 tice has been described for the head protein of T-even phages (Yanageda et a l., 1970). Bacterial viruses are not known to produce inclusion bodies. Among the nuclear viruses which are occluded [some adenovirus types, NPV, measles (Davis et a l., 1972)], only NPV has the protein crystalline lattice. In addition to the nuclear bodies containing the virus, proteineic inclusions (fusiform and spindle-shaped bodies) have been found in cytoplasma of NPV-infected c e lls (Huger and Kreig, 1969; Cunningham, 1970a, b ) . Nuclear inclusions associated with tobacco etch virus (plant virus) are composed of layers of protein to form lamellae (McDonald and Hiebert, 1974); other nuclear inclusions bodies are non-specific in nature.

Serology Nucleocapsids show serological relationships to other nucleocapsids, NPV or GV, in the same genus. Insect polyhedral proteins are antigeni- cally similar based on complement fixation studies of virus with its polyhedral proteins or immunodiffusion of polyhedral proteins alone (Krywienczyk and Bergold, 1960, 1961). Recently, by immunodiffusion, the presence of an antigen common to NP virions and their polyhedral proteins was first demonstrated (Scott and Young, 1973). A similar antigen has also been reported in granulosis virus (Longworth e t al_.,

1972); a protein on the surface of the envelope was also present in the inclusion envelope. Groups of viruses which are morphologically very similar within groups and have been antigenically typed are picomavir- uses, adenoviruses, herpesviruses, and poxviruses. Some groups of vertebrate viruses have different antigens which may be due to variation in their capsid structures (Bernard et a l., 1974). 14 Infection and Replication

In lepidopterous insects, NPV inclusion bodies are formed in the nuclei of epidermal cells, tracheal fat and blood cells; in hymenopterous insects the polyhedral formations are in the nuclei of epithelial cells of the midgut; in the dipteran insects, the nuclei of blood and fat cells. Cynological changes reported in dipteran cells were an increase in the siz e of chromosomes, accompanied by increased DNA content and concentra­ tion of ribosomes in cytoplasm (Morgante et a l., 1974) . Autoradiographic studies showed an increased DNA synthesis which is among the early effects of oncogenic viruses in mammals (Howaston, 1971). During early infection of NPV, a granular chromatin nuclear mass appears in infected cells as the nucleus grows larger (Smith and Xeros, 1953; Yamafugi, 1952). Other rod-shaped viruses with nuclear assembly are insect rhabdovirus (sigma and three others) and one plant rhabdovirus. In NPV infected cells, the whole cell is destroyed by the release of fully grown viral inclusion bodies and all cellular contents are emptied into the hemolymph (Bird,

1959; Bird and Whalen, 1954; Harrap, 1972a). Other viruses associated w ith hemocytes are an entomopox (Federici e t a l . , 1974; S toltz and

Summers, 1972), iridescent virus (Leutenegger, 1967), leukocyte replica­ tio n of herpes and vaccinia (Nahmias e t a l ., 1964; M iller and Enders, 1968). Colorado tick fever virus attaches to red blood cells (Emmons et a l., 1972), and avian erythromyeloblastic leucosis (Kit and Dubbs,

1969). In cells newly infected with NPV, a virogenic stroma is closely associated with the development of Feuglen-positive rods within vescicles of the stroma (Harrap and Robertson, 1968; Smith and Xeros, 1953; Xeros, 15 1955, 1956). In the formation of the iridescent virus capsids there are two interpretations of assembly which have been speculated to actu­ ally be more like NPV (Bird, 1961; Kelly, 1972; Smith, 1967; Xeros,

1964). The polyhedron is the form which transmits the virus infection from adult to larvae in nature. Larvae feed on foliage which is contaminated with polyhedra from previously infected and degenerated larvae. The alkaline content and enzymes within the larval gut apparently dissolve the protein matrix of the polyhedron, releasing virus particles (Harrap, 1973). The suggested pathway of v irions is from the gut lumen to n u c le i. They appear to be transported in vescicles, replicated without polyhedra production and ultimately released into hemolymph (Harrap and Robertson,

1968; Hunter et a l., 1973). Another possible pathway is via the inter­ cellular spaces between columnar cells to basal lamina and then into the hemocoel as suggested for the invasion of a granulosis virus (Tanada and Leutenegger, 1970). Goblet cells of the gut have not been reported to contain NPV particles. During the infectious process in Lepidoptera, the envelope of Baculoviruses appears to fuse with the cell membrane of the microvilli of the gut cells or plasma membrane of susceptible cells (Kawanishi et a l., 1972; Robertson et a l., 1974; Summers, 1969, 1971). Phagocytosis and viropexis may also be operative functions during particle uptake (Granados, 1973; Kislev et a l., 1969) as in granulosis infections (Summers, 1969). It is reported that the envelope is necessary for the uptake of virus by midgut epithelial cells (Harrap, 1970; Summers, 1971), but not necessary for uptake by pupal hemocytes (Kawarabata, 1974; 16 Khosaka and Himeno, 1972).

Host Responses NPV infection in susceptible hosts typically changes the behavior of infected larvae; i.e. decreased feeding and climbing to the top of vege tation (Aizawa, 1963; Smith, 1967; Tanada, 1967). In contrast, active feeding at early stages of granulosis infections is reported (Whitlock, 1974). Normally clear hemolymph turns milky-white in NPV infection and liquified body contents are released by rupture of flaccid integument

(Bergold, 1953). Prolonged larval development and moulting deviations have been frequently observed in virus infections (Federici et a l., 1974; M orris, 1970; S ta irs , 1965a; Tanada, 1954; Vago, 1951, 1956; Wigglesworth

1965). Cabbage looper larvae infected with CPV pupated as much as 5 days la te r than healthy larvae, had decreased ra te of emergence and in ­ creased moth deformities (Vail et aJL, 1969). Precocious development of adult characteristics in virus-infection have been described in Lepidop- tera based on histochemical studies of neurosecretory products (Morris,

1970). A less concentrated viral suspension extends the incubation period both in the field and in the laboratory (Aizawa, 1963; Hall, 1957). The larval mortality of G. mellonella infected with NPV was directly related to the size of virus inoculum, however, mortality in post larval stages was not predictable (Stairs, 1965b). NPV is occasionally reported in post-larval stages (Martignoni, 1964), whereas, CPV is commonly found in pupae and moths (Smith, 1967). CPV infections do not necessarily follow a direct dosage-mortality relationship (Magnoler, 1974) . Other factors affecting host susceptibility to NPV and GV are age and weight 17 at time of initial infection. The high susceptibility of young larvae and larvae of low weight has been demonstrated for several species

(Tanada, 1956; Stairs, 1965a; and others). Intraspecific variations in insect susceptibility to viral disease may be mainly genetic variation of the host (Martignoni and Schmid, 1961;

Stairs, 1965b, 1968). Differential susceptibility of strains of hosts to CPV and GV have been reported (Aruga and Watanabe, 1964; David and

Gardiner, 1960; Sidor, 1959). Induced infections have been reported from cold treatment (Ayuzawa, 1961) and chemical treatment (Aruga, 1968) . Susceptibility to virus infection differs in studies selected by induced infections compared to those selected by infection of ingested virus (Aizawa and Kurata, 1964). Changes in the blood of G. mellonella infected with NPV and in starvation include a decrease in the total number of circulating cells and significant change in the number of spherule cells (Shapiro, 1968) . In NPV in fe ctio n , spherule c e lls increased in numbers whereas in starv a­ tio n they decreased .in nuniber. Decreased hemocytes also occur in other insect diseases, e.g., rickettsial disease (Steinhaus, 1963). Replica­ tion of NPV is reported to be mostly in plasmatocytoids, some in granular hemocytes and oenocytoids, and none in adipohemocytes of cotton worm

(Kislev et a l., 1969). Other cell differentials have been reported in v ira l disease (Shapiro, 1967; W ittig, 1966). Phagocytosis of foreign particles by blood cells is recognized as an important defense mechanism in in sects (S a lt, 1970). Several studies provide indirect evidence of increased NPV susceptibility of larvae in­ jected with latex particles, ink, or carmine (Cameron, 1934; Harshbarger and Heimpel, 1968; Stairs, 1964a): uptake of particles, acid phosphatase activity (Lai-Fook, 1973; Neuwirth, 1973). Phagocytic defense is depen­ dent upon activation of hemocytes capable of phagocytosis (Kreig, 1963;

Salt, 1970). Injection with foreign agents elicits an initial increase in the number of total phagocytic cells, followed by a decrease in the total number of hemocytes over the duration of the disease process

(Shapiro, 1967). The rate of response of wax moth larvae to injection of various pathogenic factors is dependent upon the dose and kind of pathogenic agent (Seryczynska et a l., 1974; Stairs, 1965b). Starvation of wax moth larvae increased the number of degenerating cells but did not increase the number of spherule cells as in NPV infection (Shapiro, 1968) and in injections with latex p a rtic le s and India ink (W ittig, 1966) . Phagocytes may increase in number during moulting and metamorphosis (Wigglesworth, 1965). A recent biochemical study compared phagocytic cells (insect hemocytes) to mammalian leukocytes (Anderson et a l., 1973). Orthopteran insects may have blood-forming tissue analagous to vertebrate hemopoietic organs, but none has been reported fo r Lepidoptera (Hoffman,

1973). The levels of amino acids and to ta l proteins change in hemolymph of Heliothis larvae infected with NPV (Shapiro and Ignoffo, 1971; Young and

Lovell, 1971). Within the first 24 hours of infection, existing condi­ tions are hyperaminoacidemia and hyperproteinemia, whereas 96 hours post-infection conditions are hyperaminoacidemia and hypoproteinemia. Contradictory reports were apparently related to the time of the obser­ vations during the infectious process. It is suggested that changes are related directly to assembly of viral protein (Hay et a l., 1968; Joklik, 1965; Kaplan and Ben-Porat, 1968; M orris, 1968; Salzman e t a l ., 1963) and/or inclusion body protein (Morris, 1966; Smith and Xeros, 1954). In early infection protein synthesis increases in hemolymph as well as in fat body (Shigematsu and Noguchi, 1969a,b,c). Several studies sug­ gest that fat body is the primary site of hemolymph protein synthesis

(Hill, 1965; Price and Bosnian, 1966; Shigematsu, 1958). Latent Virus Occult viruses of NPV have been reported (Himeno eit a l., 1973) however there was no te s t of polyhedral development a fte r 6 days with no temperature change. It is suggested that the occult viruses are in a special physiological inactive state and are the non-infectious form (Aruga, 1963). Activation of occult viruses has been by stress factors (Steinhaus, 1958; Aruga, 1963; Smith, 1963) and by feeding a foreign virus (Longworth and Cunningham, 1968; Smith, 1963; Smith and Rivers,

1956). The inducing agent is thought to be the polyhedron protein of the nuclear polyhedrosis. In a synergistic situation of NPV and GV of arrnyworms, it was the granulosis capsule protein which was thought to be a factor of inducing increased levels of infection (Tanada, 1968). A provirus hypothesis has been suggested for the sigma virus of

Drosophila since no slow-growing sigma was found and host response was not thought probable (Seecoff, 1962). Other viruses showing la te n t te n ­ dencies are herpes, shope in humans, and temperate phages (Davis et al_.,

1972).

Host Range In vivo host range of insect pathogenic viruses is very specific to the class Insecta (Ignoffo, 1968, 1973). The NPV has been found in 20 lepidopteran and hymenopteran insects (Bergold, 1963b; Ignoffo, 1968) with a few recent reports of a baculovirus in Diptera and Orthoptera ([gnoffo and Hink, 1971). The host range of granulosis virus, the other baculovirus, is less extensive than NPV. Cross transmission of an NPV to an alternate host (terminology of primary and alternate hosts are from Bergold, 1953) is usually limited to a genus within a family that has been cross-transmitted to an alternate host in another genus (Ignoffo, 1968). Granulosis viruses have not been demonstrated to cross orders. Methods of testing have been per os introduction of polyhedra or chemically-released particles, or intrahemocoelic injection of chem­ ically released particles. In vitro specificity of insect viruses is dependent upon organ/tis­ sue susceptibility, rather than whole organ specificity. "The prob­ ability of specificity existing in a given system decreases as one sim­ plifies the host-recipient and/or the virus-donor system" (Ignoffo, 1968); therefore, primary explants and cell lines may support virus multiplica­ tion not found in the whole organism (Vail et a l., 1973). Occluded in­ sect viruses have been reported propogated in insect explants, in tissue or in cells, and in continuous cell lines (Faulkner and Henderson, 1972; Goodwin et a l., 1973; Vail et a l., 1973; Ignoffo and Hink, 1971; Knudson and Tinsley, 1974; Sohi and Cunningham, 1972) . Several species of NPV have been replicated in tissue culture (Raghow and Grace, 1974). There are no reports of successful cross-transmission in vivo or in vitro of the NPV of G. mellonella.

Infectivity The inocula used for in vivo infectivity test of NPV viruses have 21 been varied. PIB's (polyhedral inclusion bodies) have been spread onto

food, or force-fed to healthy host larvae (Chautani, 1968; Gudauskas and Canerday, 1968; Harrap, 1970; Ignoffo, 1965; Ignoffo and Garcia, 1966;

Magnoler, 1974; Martignoni and Schmid, 1961; Paschke e t a l . , 1968). Chemically released infectious particles have been fed to larvae

(Gudauskas and Canerday, 1968) or injected into the hemocoel o f pupae

(Khosaka and Himeno, 1972). No infections have resulted from the in je c ­ tion of washed virus inclusion bodies (Bergold, 1953). Insect cell lines support the replication of arboviruses, wound tumor virus, and iridescent viruses (Grace, 1969), but are only recently reported to

also support growth of an occluded virus NPV (Goodwin et a l., 1970; Ignoffo and Hink, 1971). Polyhedra were seen in the cell nuclei within 3 days; cell lysis was nearly complete by the 8th day (Faulkner and Henderson, 1972; Goodwin et a l., 1973; Ignoffo et a l., 1971; Sohi and

Cunninghara> 1972; V ail elb a l . , 1973). Infectivity of virus particles produced in cell cultures have been tested by injection of infected culture medium and/or infected culture

cells into larval hemocoel (Goodwin et a l., 1970; Henderson et a l., 1974; Sohi and Cunningham, 1972). Injections of the supematent from hemolymph

of NPV or GV infected larvae demonstrated the presence of infectious particles in inocula (Barefield and Stairs, 1969; Dimmitt, 1968). A more recent study reported that the enveloped .and the non-occluded enveloped virions from polyhedra were almost equally infectious by hemocoelic injections in to silkworm (Khosaka and Himeno, 1972). Unenveloped nucleo- capsids from hemolymph were nearly 100X more infectious by hemocoelic injections into silkworms; the oral route of infection may be different 22 (Kawarabata, 1974). Other reports suggest a difference between occluded and non-occluded virions of NPV. In addition to a different affinity for tissue culture, paper chromatography shows a difference in patterns (Vaughn, 1965). An EM study supports these differences (Kawarabata, 1974) . Filtered (450 nm) infected hemolymph was infectious for tissue culture

(Sohi and Cunningham, 1972).

The smallest infectious particle of granulosis virus may be a unit less than an entire intact virus particle (Summers and Paschke, 1970; Zherebtzova et a l., 1972). The infectivity of small spherical particles of NPV (20-26nm) is disputed (Aizawa, 1963; Kreig, 1963; Khosaka and Himeno, 1972; Dimmitt, 1968). RNA extracted from NPV infected tissues of

G. mellonella induced formation of polyhedra in healthy recipients (Kok et a l., 1967) . This development was not attributed to latent viruses. Physiochemistry NPV polyhedra are insoluble in water, alcohol, ether, chloroform, benzol, and acetone and are not decomposed by proteolytic bacteria and fungi (Jaques and Huston, 1969). The silicon content may contribute to polyhedral resistance to breakdown by bacteria (Jaques, 1967) . Formalin treatment of PIB reduced viral activity (Ignoffo and Garcia, 1966). In contrast, polyhedra of cytoplasmic polyhedrosis virus (CPV) are susceptible to bacterial putrefaction (Hills and Smith, 1959). An optimal recovery of baculovirus virions has been reported using 0.05M

Na2C03 for 30 min. to 1 hr. (Harrap and Longworth, 1974), first described by Bergold (cited in Bergold, 1963b). Dissolution of polyhedra in alkali

30% more concentrated than needed to release rods from polyhedra may disintegrate the rods (Bergold, 1963b). Onset of polyhedra dissolution 23 varies with the host species (Smirnoff, 1966). Bombyx mori disintegra­

tion starts at O.OOZSM^CO^ (Bergold, 1963b). Divalent ions, e.g., Ca++,

Mg++ were reported disruptive to the virion envelope (Bergold, 1963b; Shapiro and Ignoffo, 1969) but was not reproducible upon rete stin g (Kawanishi and Paschke, 1970; Summers and Paschke, 1970). Information

on physiochemical properties of NPV is available (Aizawa, 1963; Benz,

1963; Bergold, 1963b; Smith, 1967). A recent evaluation of p u rificatio n methods of virus p a rtic le s and nucleocapsids of granulosis and nuclear polyhedrosis viruse particles recommends the use of sucrose gradients after a limited period (30 min.- 1 hr.) of alkaline treatment (Harrap and Longworth, 1974) . Even after differential centrifugation, virus particles may be in various forms

(Harrap, 1972b; Kawanishi and Paschke, 1970; Summers and Paschke, 1970). The duration of dissociation treatment of polyhedra showed a signi­ ficant decrease in infectivity between 0.5 and 25 hours of NPV of H eliothis and Trichoplusia (Gudauskas and Canerday, 1968) . Data in d i­ cates the importance of maintaining NPV near neutrality for maximum in­

fectivity (Gudauskas and Canerday, 1968; Ignoffo and Gracia, 1966; Kawanishi and Paschke, 1970). Phosphate did not effect the infectivity of Heliotis zea NPV (Gudauskas and Canerday, 1968). Inactivation of various picomaviruses by alkaline treatment has been widely studied. Under certain conditions, polio loses its genome (Berghe and Boeye, 1973);

several coxachieviruses become unable to attach to susceptible cells (Cords e t a l . , 1975). Lack o f attachment is due to a sp ecific lo ss of a small polypeptide at low ionic concentration. Disruption of polio- virus increases with increased temperature (32.5-40), increased pH and 24 increased NaCl (up to 0.3M) (Boeye and Elsen, 1967). Low ionic concen­ tration does not affect polio viruses.

Degradation by nucleases is usually a part of purification proce­ dures. Recently, a report was made of the use of DNase to decrease the infectivity of chemically-freed granulosis virus particles (Summers and Paschke, 1970). A study on poliovirus demonstrated that viral nucleic acid could be protected from nuclease by the capsid, but loses 90% of its infectivity from alkaline treatment (Berghe and Boeye, 1973).

Storage Occluded insect viruses are known to retain infective activity for long periods of storage. Refrigerated, infectivity has lasted 20 yrs. in a sealed vial (Steinhaus, 1960). Polyhedra stored at room temperature fo r 8 months were infectious (Hukuhara and Namura, 1972). At low temper­ atures (0-20°C) NPV retained infectious activity up to 18 months in water

(McEwen and Hervery,1959) . -A titration of polyhedra stored 6 months a t 0°F showed no decrease in infectivity (Getzin, 1962). Filtered (0.8u) NPV infected hemolymph stored in liquid nitrogen for 13 months was in­ fectious to tissue culture (Vaughn, 1972). After 1 year of storage at 0-3°C, a granulosis virus decreased in infectious activity (David and

Gardiner, 1967; McEwen and Hervery,1959); 20 days at room temperature (20°C and up) caused a decrease in infectivity (David et al_., 1971). An alkaline protease in polyhedra of Bombyx mori and G. mellonella showed decreased activity 2-3 years in cold storage within polyhedral forms (Kozlov et a l., 1975). The susceptibility of other viruses varies with the virus (Plummer and Lewis, 1965; Thome and Holt, 1974). Most viruses "in infected tissue are not harmed by freezing and thus are 25 prefrentially stored and accumulated at low temperatures" (Fraenkel-Conrat,

1969). An exception to this is TMV (purified). Virus particles, once released from the polyhedra are unstable for storage purposes, especially below temperatures of 0°C (Harrap and Longworth, 1974). Insect hemolymph was a better stabilizer than gelatin or glucose for granulosis virus of cabbage worm (David et a l., 1971); glycerol re­ duces c e ll damage up to 6 months a t -29°C (Tressler

(Sutter, 1972) . Newly replicated unpurified polyhedra were 4 log dilu­ tions more infectious than polyhedra stored at -30°C (Ignoffo, 1964a, b). NPV can be produced from generation to generation in laboratory stocks or in field infections. Field conditions include fluctuating temperatures involving m ultiple freezes and thaws (Clark, 1955) and UV exposure. Multiple thaw conditions make polio virus susceptible to urea and guanine (Cooper, 1962; Drzeniek and Bilello, 1972) which weaken the protein bonds (Kauzman, 1959). Polyhedra and granulosis are both re­ ported to be sen sitiv e to UV sunlight (David e t a l . , 1971; Gudauskas and

Canerday, 1968). NPV in fie ld cadavers showed no change in virulence over 14-18 years from generation to generation (Bird, 1955). Serial passage of NPV in cell lines showed increased infectivity of NPV

(Shapiro and Ignoffo, 1969). 26

MATERIALS AND METHODS

Virus.--The nuclear polyhedrosis virus used in these studies infects G. mellonella in nature. The virus source was obtained from a refriger­ ated sedimentation of sucrose-gradient-purified polyhedra remaining from the putrifaction of several infected larvae in water. The original strain was obtained from the laboratory of Dr. Bird in 1965 via Dr. G. R. Stairs. Occluded virus particles were released from inclusion bodies by a mild alkaline treatment. Non-occluded infectious virus particles were obtained from the hemolymph of fresh infected larvae by maceration with a glass rod in a 15ml centrifuge tube containing 5 or 10ml water. Frozen infected larvae were placed into individual gelatin capsules and kept in P e tri dishes marked with date, experiment number, and other pertinent information. The freezer used for storage was a horizontal type so that frequent opening and closing did not affect temperature at the level of storage of the virus-infected cadavers. For testing, fro­ zen larvae were thawed slowly at room temperature.

Laboratory rearing of host.--Stocks of G. mellonella were established from larvae originally in the Ohio area. Larvae were reared on modified n u tritiv e medium (Waterhouse 1959; S ta irs , 1965c) , prepared from an auto- claved mixture of 90ml raw honey, 45ml gycerol, and 45ml water. Just before use, 3-5gm of yeast extract and 1 box (227gm) Pablum (Mead Johnson and Company, Evansville, Indiana) was added to the mixture. Glass con­ tainers with screened screw tops were filled one-third full with this medium and used as rearing containers. Cultures of insects were 27 maintained by removing slightly anesthetized (CO 2) newly emerged adults and placing them into a Petri dish or similar plastic container with pleated wax paper to allow oviposit ion and adhesion of eggs. Several masses laid within a 2 -day period would produce a new generation in another breeding jar. Decreased moisture delayed development or com­ pletely dried out eggs. Test larvae, as well as controls, were selected from a single gener­ ation (same age in days) and medium size (approximately same in overall size). Random assortment into groups of 5-10 larvae provided test groups for the various dilutions of virus suspensions and controls. Test and control larvae were placed into nutrient medium in labeled Petri dishes (60 x 15mm). Each dish contained 1-5 larvae and was incubated on a near normal (12-12) light-dark regime at approximately 27°C. Enzymes. - -Deoxyribonuclease-I (Bovine Pancreas, Sigma Chemical Co.,

Mo.) and protease-free ribonuclease-A (Bovine Pancreas, Sigma Chem.) were used at concentrations of 100 and 200 ug per ml of virus suspension. Enzyme solution of DNase in water was added to a substrate solution of

1.0M acetate b u ffer, pH 5 .0 , 0.1M MgSC>4, plus a virus suspension. RNase (4.6mg/ml) was dissolved in a 1.2M Na^PC^ buffer, at pH 6.4 with NaOH

(Gomori, 1946). Alkaline solution.--A modification of Bergold's (1958) salting out of proteins was used to release occluded virions from inclusion body proteins. A solution of 0.01M Na 2C0 3 and 0 .0 5M NaCl has a to ta l ionic strength of 0.08 (Bergold, 1963). The (total) ionic strength (I) is ex­ pressed as one-half of the ionic contentration according to Lehninger

(1970). 28

The pH values were determined with Short Range Alkacid Test Papers (Fisher Scientific, Pa.) or an electrode-type pH meter (Beckman Instru­ ments, Fullerton, Ca.). Centrifugation. - -Macerated infected larvae in water were centrifuged at 3,000 rpm for 30 min. in a refrigerated Sorvall centrifuge. Virus suspensions were ultracentrifuged in a refrigerated Beckman at 50,000 rpm for 4 hrs. in the #50 rotor. Upon removal from the centrifuge, lipids on

the top were carefully removed by using a Pasteur p ip et, or the underlying supematent was withdrawn directly without disturbing the lipid layer on

top. M icrofiltration. - -Virus suspensions were passed through Millipore (M illipore Corp., Bedford, Mass.) f ilte r s of HA.450nm, GS220nm, VClOOnm, or VM50nm pore siz e . F ilte rs (mixed e ste rs of cellulose) were mounted

in stainless steel Swinny filter holders and autoclaved in brown taped bags. Filter units were kept in these bags until needed. Microin j ection. - - Inocula of virus suspensions were injected into the hemocoel of healthy larvae with a 1-inch #27 disposable needle (B-D‘ Yale)

on a Hamilton microapplicator. Glass syringes were used. Virus suspen­ sions were injected behind the prolegs on the left lateral line of the

larva, with the needle angled posteriorly. Light microscopy.--Hemocytes were examined for the presence or ab­ sence of replicated polyhedra on a Leitz phase microscope. A drop of hemolymph provided material for a wet preparation. Polyhedra counts of refrigerated stock were counted in a Petroff-Hausser double-chambered hemocytometer. 29 Electron microscopy. —Ultracentrifuged virus sediment was examined with an electron microscope (RCA, 3G) . Samples were centrifuged and negatively stained with neutral 2% phosphotungstic acid. Virus prepara­ tions were then sprayed onto carbon-coated EM copper grids with a Pellco vacuum spray gun from a distance of approximately 3 feet. Sucrose gradient. - - Gradients were prepared according to the method of Barefield and Stairs (1969) using a commercial grade of sucrose. Ten percent intervals, 5% to 451 sucrose by weight, were layered by adding increasingly heavier solutions under previous solutions in the gradient. A centrifuged virus pellet resuspended in 0.5ml balanced salt solution (PBS) was layered on top of the gradient and centrifuged 4 hours at 30,000 rpm in a SW#39 rotor of the Sorvall centrifuge. Titration of virus.--Viral suspensions were diluted in 10-fold mul­ tiples with double distilled water. Aliquots of lOul were injected into the hemocoel of healthy wax moth larvae. For each dilution 5-10 larvae were used to assay the activity of the inocula. Probit regression lines were calculated from weighted values of total mortality according to

Bliss (1938) fo r small numbers. LD50 values were estimated using loga­ rithmic-probit analysis (Bliss, 1938). Sampling hemolymph and counts of infected cells.--Periodic examina­ tion of hemolymph was made, noting the absence or presence of polyhedra and stage and time of death of each mortality. Drops (a single drop, preferable) of hemolymph obtained from a slight puncture with an insect pin (alcohol flamed) to a location opposite the site of the original in­ jection, i.e., the right lateral side behind the prolegs. With few

infected hemocytes, (less than 101) 1 ,0 0 0 hemocytes were counted and examined-; otherw ise, counts -of - 100 -hemocytes-were- made-. 30 Expression of virus concentration.--Virus concentration of inocula prepared by the release of occluded particles from refrigerated polyhedra were calculated to obtain the final concentration of the original poly­ hedra count. Inocula prepared from whole macerated larvae, were expressed in terms of dilutions of the infected larvae, i.e., parts of infected lar­ vae per ml of inoculum. 31

RESULTS

Effect of storage and serial passage. Storage.--The infectious activity of free virions that had been fro­ zen in infected larvae was determined by bioassay. Assays were based upon the appearance of newly replicated polyhedra in the nuclei of hemocytes and further verified by the capacity of infected hemolymph to cause rein­ fection by injection. No mature polyhedra were observed in hemocytes and no deaths occurred when larvae were injected with virus that had been frozen in live larvae or cadavers for 1 year or longer (Table 6 ) , Inoculations prepared from infected larvae th a t had been frozen 1-2 months produced consistent infections. Mortality occurred in all stages of host development. Mortality of 100% and a rapid rate of polyhedra development in larvae resulted from injections of a similar concentration of infected larval parts from freshly infected larvae (no alkaline treatment). This suggests that freezing is detrimental to free, non-occluded virus particles. It appears that increased numbers of larvae with polyhedra is re­ lated to increased concentrations of infected tissue, however, the status (live or dead) of the infected larvae when stored may influence the amount of infectious activity present. No polyhedra were formed in larvae injected with a concentration ( 0 .2 infected larval parts/ml) of alkaline-treated (1/2-hr.) virus that had been frozen (-10°C) in cadavers for 32 months (Table 7). With twice the concentration (0.4), polyhedra developed in 87% of the injected larvae with virus from larvae that had been frozen alive. 32 Other factors influencing the infectious activity of frozen infected hemolymph were the duration of storage and the ionic concentration of the alkaline solution. It was found that the optimal ionic concentration to release refrigerated occluded particles was 0.05M (Table 8). A solution of 0.05M Na2C0 2 -NaCl with NPV frozen 30 or more months did not produce any frank infections. Virus frozen 30 months increased in infectious activity from no infections with 0.05M alkaline solution to 931 infection using the 0.10M alkaline solution (Table 9 ). Virus frozen 12 months and incubated with 0.10M alkaline solution produced 7% infectivity. These data suggest a difference in the biochemistry or structure and function of the virus particles and/or polyhedra with increased frozen storage time. There was no noticeable relationship between the length of storage and infectivity of polyhedra stored in aqueous solution (Table 10). Re­ frigerated polyhedra were tested at various times throughout a period of five years. Mortality and polyhedral development in hemocytes occurred in 60-1001 larvae injected with alkaline-treated virus particles. Prolonged alkaline treatment.--Infected cadavers stored 2 1/2 years at -10°C decreased in infectivity when treated with alkaline solution for 20 hours. Infectivity of 92% (1/2 hr. 0.10M alkaline treatment) decreased to 17% by the additional 19 1/2 hours of alkaline (0.10M) incubation

(Table 9). Use of 0.05M alkaline solution resulted in no polyhedra pro­ duction in either of two test groups of 30 larvae each. In contrast, in cadavers stored 12 months at -10°C the additional incubation period (using 0.10M alkaline solution) increased the infectious activity from 7% to 69% (Table 9). In most cases, infection was verified by the 33 presence of polyhedra. Pupae died without polyhedra. Prolonged alkaline treatment did not decrease infectivity and mortality (Table 9). Incubation with sodium chloride.--The effect of prolonged exposure of refrigerated occluded virus particles and freshly-replicated non­ occluded particles to NaCl was tested. The refrigerated occluded parti­ cles released at the end of the incubation period by Na 2C0 2 (0.0025M for 1/2 hr.). Larvae injected with NaCl-incubated released occluded parti­ cles did not produce polyhedra but did have high mortality rate. Agar plates showed no bacterial contamination. Occluded particles exposed to

Na2C0 j-NaCl for 24 hours produced polyhedra in at least 50% of injected larvae with the same high mortality as the salt-incubated virus suspen­ sion Table (11). The salt treatment appeared to interfer with virus replication since 1/2 hr. ^ 2(1)3-NaCl routinely produced high levels of infection. Most deaths from NaCl-incubated suspension occurred in the pupal stage of host development, supporting the role of NaCl incubation in subsequent interference of virus development.

Infectivity of non-occluded virus particles was the same for NaCl- incubated as for ^ 2^ 3-NaCl incubated. Death was more rapid in both of these test groups than in the groups of larvae injected with untreated non-occluded virus. This indicated a higher level of virulence in the incubated virus suspensions. Salt did not affect the infectivity of non-occluded virus particles (Table 11). Serial passage.--NPV was consecutively passed through 14 host gener­ ations. Filtered infected hemolymph was used for each passage following the first. The original infection was produced by injection of alkaline- released virus from polyhedra in aqueous solution at 8°C. Passages were 34 identified by source and treatment. (Source is described by the filter pore size and dilution of material injected into source larvae to produce new infection; the concentration of infected material prepared as new inoculum and the filtration-dilution treatment describes the treatment of a given inoculum.) Larvae used as a source of inoculum were those which exhibited mature polyhedra in hemocytes as evidence of virus in fectio n . Virus suspensions were filtered before the preparation of log-dilutions.

Suspensions ranging from 1.3 x 10"^ to 1.8 x 1 0 infected larval parts/ml were filtered through a sequential series of filters. All preparations were filtered through 450nm pore size. Some preparations were further filte re d through 220 and lOOnm. V iral a c tiv ity and ra te of development were relatively constant throughout the passage series (Tables 12 and 13;

Figs. 1-5). Non-occluded particles in infected hemolymph remained infectious through various filter treatments as well as several other treatments, including 451 sucrose gradient and 9 days in v itro a t 8°C in passage numbers 3 and 12, respectively (Table 12). The decreased v iru s a c tiv ity in passages 6 and 9 (Table 12) are not explainable at the present time. Virus and polyhedra replication and development in hemocytes. A group of 30 mature larvae were injected and monitored individually for several days. Within one day, most of the larvae (25/30) contained a few hemocytes (0.5, range 0.1 to 1.0%) with mature polyhedra in their nuclei (Table 14, A). Some of these larvae contained only one infected cell per 1000 while others contained as many as 10 per 1000. On the second day 12 percent (range 5 to 30%) of the hemocytes contained 35 polyhedra and this increased to 60 percent (range 20 to 901) on day three Some of these larvae died on day five and all were dead by day six.

In this same group of larvae, some (5/30) did not have mature polyhedra in their hemocytes until the second day after they had been injected (Table 14, B) . Otherwise, the pattern of hemocyte infection was similar to that in the rest of the larvae in this group.

Similar monitoring of hemocytes was done on another group of 38 larvae following the injection of a less concentrated virus inoculum

(Table 14, C) . One day after injection, only two larvae of the 16 (12%) examined contained polyhedra in a few hemocytes (0.3, 0.1 to 0.5%). On day two, 38 larvae were examined and 20 (53%) contained polyhedra in 6 percent (range 0.1 to 20%) of their hemocytes. On day three, four larvae s till did not have polyhedra in their hemocytes although 20 to 50 percent of the hemocytes of the others (34) contained mature polyhedra. The hemo cytes of these larvae, however, developed mature polyhedra and the larvae were dead on day six along with all the other treated larvae. The appearance of mature polyhedra in hemocytes was variable. De­ spite the fact that all larvae received the same virus dosage, each appeared to react differently. In some, polyhedron formation occurred in a few of the hemocytes within 24 hours, whereas in others it was de­

layed until the second or third days. Once a few hemocytes were infected the disease progresses rapidly and in a predictable pattern; larvae usually died within 4 to 5 days. In all instances, the appearance of polyhedra in the hemocytes was followed by death from the disease.

Infectious virus in hemolymph of diseased larvae. Effect of filter pore size, dilution, and serial passage. - -Relative 36 levels of infection in different passages of NPV-infected hemolymph were compared. Bioassay of infectivity determined the effects of filters, dilutions, and passage. Throughout the passage series, similar levels of infectivity were produced by infectious material. After filtration, suspensions were diluted to determine end points using different filter pore sizes. Total mortality through each of the pore sizes (450nm, 220nm, and lOOnm) were linear by a Student's t-test.

The linearity of responses was tested at t ^ 5 ^ ^ (0.20 level of significance) on a probit/log-dose basis. The rate of increased in­ fectious activity varies directly with the log-dose of infected hemo­ lymph injected. The regression lines of the different filter sizes are the same within a given experiment, and appear to have an average slope of greater than 1.0 (when log doses bracket probit 5) (Table 13, and

Figs. 1-5). Mortality doses producing 501 response were estimated from regression lines calculated from probit values of mortality data corrected and weighted. High lev els of in fe c tiv ity were obtained when virus was passed through 450nm filters. Comparison of the infectivity of filtrates from different pore-sizes within experiments revealed that the 220nm and the lOOnm pore-sizes retained infectivity present in the 450nm filtrates.

There was a difference o f 6 log units between the activity of prepara­ tions treated by the 450nm filter and the 220nm filters. More than 99.99% of the infectivity present in the 450nm filtrate was held back by the 220nm f i l t e r . Differences between the LD^g levels of infectivity in 220nm and lOOnm filtrates were not consistent, varying from zero to 5 log units 37 less infectivity in the lOOnm filtrate. To examine this inconsistency, three aliquots of a given virus preparation were filtered through a lOOnm separate while a fourth aliquot was filtered through a 220nm pore- size. There was a consistent difference of a least 3 log units between the two f i l t e r size s. The lOOnm f ilte r s retained 60-751 of the infectious material present in the 220nm filtrate. Virus suspensions filtered through 50 nm Millipore did not produce polyhedra, but did have mortality not seen in controls. In one experi­ ment, three of the dead larvae injected with 50nm filtrate were prepared as injection material. Infected hemocytes were observed in 2 out of 10 larvae at 4 x 10 and 10 larval parts/ml of inoculum. No infection was produced at higher dilutions of the inoculum. Electron microscopy observations.--Electron micrographs of virus that had passed through a lOOnm filter showed bent rod-shaped particles

(Fig. 6 and 7) . These particles were broken and/or bent whereas parti­ cles that passed through larger pore sizes (450nm and 220 nm) were straight. Particles were 38-40nm in diameter and 100-400nm long. Observed rods were without membranes.

Common to all filtrates was the presence of small particles 7-9nm in diameter (Fig. 6 ,c and 7,C). These particles appear to be composed of several subunits, 4-6 in number, each 2-4nm in diameter. Larger particles appeared to have more of these smaller subunits (Fig. 6,b;7,b). Effect of DNase on infectious activity.--To detect the presence of infectious DNA in a form susceptible to DNase, filtered (450nm) hemo- lymph from infected larvae was incubated with DNase and bioassayed. Larvae injected with DNase-treated hemolymph showed higher rates of in­ fections than the controls. Mature polyhedra were observed in 90-100% 38

of larvae injected with virus (dilutions of 10 and 1 0 “^® infected

larval parts/ml inoculum) plus DNase. Infected hemolymph without DNase produced infections in 30 to 50% of injected larvae (Table 15). There was no difference between lOOug and 200ug DNase based on the tested levels of infected hemolymph.

The in itial rate of polyhedra development varied with DNase. With lOOyg DNase, two-days post-injection, 53% of the larvae had at least 30% of hemocytes infected. Without DNase, infected hemolymph produced

30% or more infected hemocytes in 87% of the injected larvae (Fig. 8). In larvae injected with enzyme-incubated virus, there were several modes in the distribution of percent infected hemocytes, whereas the infected hemolymph alone had a single mode of 30%. A ll deaths in enzyme te s t larvae were confirmed viral infections. The susceptibility of released occluded infectious particles of NPV to DNase was tested. Polyhedra stored 1-5 years at 8°C in aqueous so lu ­ tion were incubated with alkaline solution (pH7.5) for 1/2 hours. Larvae injected with alkaline-released virus incubated with DNase developed less infection than those injected without DNase incubation. Injection of alkaline-released particles plus lOOyg DNase produced 6 deaths in 30

te s t larvae, compared to 100% mortality in larvae injected with virus

alone. Twenty-three of these larvae had confirmed virus infection, but no polyhedra were observed in larvae dying from injections of enzyme- incubated virus. Disease development varied with the enzyme treatments to released occluded virus particles. Mortality of larvae injected with DNase plus 39 alkaline-treated virus was mostly in post-larval stages of host develop­ ment, whereas alkaline-treated virus alone had 70% of its mortality in

the larval stage (Table 16). Acetate buffer (pH 5.0, 1.0M with 0.1M MgSC^) injected into 20

larvae as a control, produced mortality similar to that in the enzyme -

treated group; 4/20 and 5/30, respectively. This suggests that, while the buffer injections do not produce polyhedra, there is a lethal effect without the enzyme action. Effect of RNase on infectious activity.--To determine the effect of RNase on infectious a c tiv ity of infected hemolymph, RNase was added to

dilutions of filtered (450nm) infected hemolymph giving final concentra­

tions o f lOOyg/ml and 200yg/ml of enzyme in each d ilu tio n . Both alk alin e- treated and non-alkaline treated infected hemolymph were tested for inhibi­

tion of infectivity by RNase. Alkaline treatments were for 1/2 hour fol­

lowing filtration and immediately prior to the addition of enzyme. RNase had an inhibitory effect on polyhedra production at dilutions of hemolymph higher than 6.5 x 10“2 infected larval parts per ml (Table

_ *7 17). At 10 log-dose of virus, half of the larvae produced polyhedra

from in je c tio n o f virus plus 200pg RNase (Table 18). At 10 log-dose

of virus, half of the larvae produced polyhedra from injection of virus plus lOOyg RNase. Without RNase treatment, virus injected resulted in

100% polyhedra production of both 1 0 and 1 0 log-doses of virus.

Agar p la te s showed no contamination in inocula. Alkaline-released occluded virus produced polyhedra in 50% larvae

a t 10 "3 log-dose of virus when incubated with lOOyg RNase (Table 18) .

Virus alone had an LD^q of 1 0 log-dose. 40 The e ffe c t o f RNase w ith its sodium biphosphate buffer (1.2M, pH

6 .4) to larvae was determined by injection of these chemicals without virus. Freshly prepared enzyme plus buffer did not cause any deaths, however, solutions of lOOpg and 200yg RNase incubated 20 hours a t 21°C produced 40-601 mortality (Table 17). Deaths occurred in all stages of host development. Prolonged incubation of RNase plus buffer appears to alter the activity of these chemicals. Effect of Alkaline treatment on infectivity of hemolymph. - -Different forms of NPV were separated from fresh infected hemolymph to compare alkaline stability of virus forms. Virus forms were separated by vari­ ation of the sequence of steps for preparation of inocula. The final concentration of NaCl and Na 2C0 j was 0.023M and 0.0024M respectively.

Each preparation was passed through a. 450nm pore-size filter. The highest level of infectious activity resulted from the inocu­ lation of infected hemolymph which had no alkaline treatment.

Lower levels of infectivity were produced by injection of alkaline- treated virus suspensions. Of the two sequences using alkaline solution, the greater decrease was in larvae injected with virus suspension which had been alkaline-incubated prior to routine centrifugation and filtra ­ tion of hemolymph. Total mortality was 50% at the highest dilution. At

a similar dilution (10 infected larval parts/ml), when alkaline- treatment followed centrifugation and filtration, mortality was 40%. Although mortality appeared similar, the rate of response varied. Alkaline treatment did not increase the infectious activity of fresh infected hemolymph (Table 19). 41

DISCUSSION

Storage arid Serial Passage.--Nuclear polyhedrosis virus (NPV) has

several form which show many sim ilarities to the various forms of her­ pesviruses. Similarities include the stability of the various foims Herpes is different in its stability of intracellular particles compared

to extracellular particles: intracellular lose 50-75% infectivity after 4 days at 4°C, whereas cytomegalovirus (a cytoplasmic herpesvirus) starts

losing (decrease to zero) infectivity at 4°C in 36 hours (Plummer and Lewis, 1965). The non-occluded particles of NPV had no infectivity after one year of storage at -10°C. This loss in activity was despite storage in i t s natural environment, the in fected larva. I t has been assumed th a t diseased larvae could be collected and stored until needed (Bird 1964; Clark 1955). The glycerol content of the hemolymph may be responsible for stabilizing infectious activity for a few months at -10°C; other­ wise, as previously reported by Shapiro and Ignoffo (1969) , non-occluded virus particles of NPV are not suitable for storage at -10°C. The same susceptibility to low temperature (-20°C) was reported for a non-occluded virus of G alleria (Longworth e t a l . , 1968). The amount of alkaline-released infectious ac tiv ity from virus th a t had been frozen decreased with time duration of storage. Another factor affecting the amount of infectious activity released was the status of the larvae when stored: alive or dead. Frozen cadavers apparently lost some infectious activity due to the release of destructive enzymes dur­ ing the moribund period of host and cells. 42 Not all polyhedron are dissolved simultaneously. The increased activity of virus released from polyhedra by prolonged alkaline incuba­ tion indicates that the rate of release has become greater than the rate of destruction by the same proportion as the increase in activity. These results are in conflict with the known detrimental effect of acid and

alkaline solutions to the secondary structure of ENA (Korriberg, 1974) , and to infectivity of NPV (Bergold, 1958). The low ionic concentration used in the present studies may be an explanation for this difference. Prolonged freezing alters the structure of polyhedron and/or the nucleocapsid. After freezing, either fewer infectious particles are released from a single polyhedra, or released particles are more sus­ ceptible to chemicals. Such altered particles may be sensitive to alka­ line solution or urea, as in poliovirus that has been frozen (Mandel.

1973). Urea is present in insect hemolymph (Wigglesworth, 1965). A similar profile of cold stability is observed in a phage of Bacillus

cereus. Inactivation of this virus is more rapid when frozen at O^C than when refrigerated (2-4°C) (Thome and Holt, 1974) . Freezer storage of infected cadavers is attractive in its ease of procedure and the pos­ sibility of preserving high levels of infectious activity, but with in­ creasing time in fe c tiv ity becomes harder to obtain. This agrees with decline in infectious activity of vaccinia virus and cabbage looper NPV at low temperatures (Sharp et a l., 1964, Thomas et a l., 1972). Bergold thought that the release (destruction) of the outer envelope contributed to decreased infectivity. Both enveloped and non-enveloped herpes are found in cellular as well as extracellular locations (Keller et al., 1970) but it is not known which form is responsible for infection. 43 An EM study showed that viTus particles producing NPV infection after passing through a lOOnm filte r were not enveloped. In addition to Kawarabata's study (1974) that the envelope is not necessary for infec­ tion in pupal hemocytes, they are apparently not necessary for infection of larval hemocytes. The stability of virulence of particles free in the infected hemo­ lymph provides a reliable method for the production of high levels of non-occluded virus particles. Serially passed hemolymph producing poly­ hedra in hemocytes also produced non-occluded virus particles. Estimated

LDgQs showed that virulence did not change. Filtration. - -The infectivity of filtered infected hemolymph depends upon the dilution effect of the filter and serial log-dilutions of prep­ aration. The linear responses to serial dilutions of infected hemolymph mean that the response is predictable. If a few injected larvae become infected at a low dose, then more w ill become infected at higher doses. The similarity of slopes of regression lines means that the particles passing through the filters of a given pore size all have similar rates of activity. A low slope of 50% or lower levels of response is a typical biological profile (Bliss, 1938). Different intercept points of the regression lines were dependent upon pore-size. The over-all effect of filtering seems to be one of dilution, since each progressively smaller pore-size retained a proportion of virus and yet each filtrate had a similar profile of activity. There is no evidence of different forms being present. Experiments in which the LD^ of lOOnm filtrate and 200nm filtrate were similar, the lOOnm could have tom. This conclusion was justified by replicate tests of lOOnm all producing a similar 44 level of activity which was decreased at least 3 log units compared to

the 220nm filtrate. Another explanation for similar slopes and separate intercepts fo r each f i l t e r pore-size is th a t equal amounts of several sizes o f

free particles were filtered through each size. Under the circumstances of easily clogged filters, a perfect distribution of all sizes through each filter is highly unlikely. The 50nm pore-size filter appears to retain all infectivity. Al­ though there were no polyhedra produced in hemocytes, some deaths and pro­ longed development did occur. However, infection was caused by passage of this newly injected hemolymph through, a 450nm filte r and injection into healthy larvae. Further investigation is needed to verify this

infectious process. It is not known what was present in the second passage that was not present in the first. Perhaps a low grade (chronic) infection existed in the larvae of the first passage. This may be similar to the high dilution doses in which larvae and pupae died without frank virus infections. Both situations prolonged host development. Perhaps non-occluded particles can be replicated without simultaneous production of polyhedra, providing the second passage with a higher concentration of non-occluded particles, thus producing polyhedra. Infections produced by the injection of particles less than the size of a single rod would provide evidence for a segmented genome. Segmented genomes are found in groups of RNA viruses: reoviruses (RCN), cytoplasmic polyhedrosis virus (RCN), rous sarcoma virus- (RHE), Newcastle Disease virus (RHE), polio virus (RCN), and influenza (RHE), (Duesberg, 1968; Hirst, 1962; Kalmakoff, 1969; Millard and Graham, 1971). Another type-of-segmentation-of virus-genome is -found in-the separate 45 encapsulation of complimentary strands of the polyoma virus (DCN). Polyoma virus i s assembled in th e nucleus as is NPV. Electron microscopy observations.--Electron micrographs showed that rod-shaped particles free in the hemolymph did not have intimate membranes. This agrees w ith the observations o f Bird and Whalen (1954), Bird (1959),

and Kawarabata (1974). The infectious activity of these particles is demonstrated by the rate (slope b) of mortality in response to virus dose. A slope (b) o f 1.0 or more describes an active in secticid e (Burges et a l., 1971); expected slopes are 1.0 to 3.0 on a probit-log scale. Free virions differed from occluded virions by the lack of menbrane, single occurrence, and variance in length. Lengths ranged from 100 to 400nm. The breaks in rods suggest that lengths shorter than 400nm may be broken-off pieces of 400nm rods. The question remains as to the infec- tivity of such broken and separated pieces. The preponderance of bent and broken-but-attached rods suggests that the broken-but-attached rods

are infectious as are the bent rods. Any of these particles, if posi­ tioned precisely by chance on end, could have passed through a 50nm pore size. No frank infections were produced from 50nm filtrates, but the possible presence of infectious material needs to be further investigated. Occasional mortality and replication of polyhedra in a further host pas­ sage implicates the passage of an occasional virus rod or an incomplete virus form through the 50nm filter. Present evidence indicates that the rods are the primary infectious particles of NPV. Interpretation of the filtration studies suggests that the small p a rtic le s (7-9nm) are not the sm allest infectious p a rtic le s. This is contrary to results of a centrifugation study (Dimndtt, 1968) and other 46 reports of stbviral infectivity. These subunits may be capsid units as suggested for adenovirus capsid (Norrby, 1969), which is not completely in contrast to the concept of them being immature virus particles (Bird

and Whalen, 1954). Virus multiplication in early disease.--The appearance of mature polyhedra in hemocyte nuclei is related to multiplication of NP virus in early stages of disease. Cytological observations describe the in­ cubation time for virus multiplication. (This inctibation time should not be confused w ithin th e same term used by Aizawa (1963) to denote the mortality time of the host.) The earliest observation of mature polyhedra in G. mellonella was at the end of the first 24 hours post-injection. This was in response to the injection with naturally free infectious particles in infected hemolymph. Polyhedra were ovserved

in silkworm larvae approximately 48 hours following the injection of an NPV (Aizawa, 1950 in Aizawa, 1963). NPVs in th e ir homologous host appear to have different incubation times for the development of mature polyhedra. There may also be a relationship between the form of the virus injected and the route that a given form must employ to produce new polyhedra. Variations within the incubation time in mellonella may be due to several factors. Incubation periods in the present studies were dependent ipon the age of larvae injected, dilution of the inocula, and

an unknown variable related to individual differences in larvae. Indivi­

dual larvae may have differences ( 1) in susceptibility among cell popu­

lation and/or ( 2) in enzyme systems contributing to virus replication within susceptible cells. Both of these factors would provide a degree 47 of resistance on the part of the host. More investigation is necessary to suggest that these individual variations are genetic rather than phy­ siological variations. The mitotic stage of susceptible cells may regulate the replication of some viruses. Metaphase cells do not support vaccinia or herpes re­ plication (Marcus and Robbins, 1963; Nahmias et aL., 1964). Susceptibility of host cells may be related to the cell type as well as the stage of cell * division. The hemocyte cell type (s) which supports the replication of NPV have not been identified. In the present study, as many as 901 hemocytes were infected with polyhedra following the injection of non-occluded virus p a r tic le s . By visu al id e n tific a tio n , Werner and Jones (1969) estim ated as high as 95-98% of hemocytes to be phagocytic in Galleria. From this it is concluded that the Npv is replicated in phagocytic hemocytes. How­ ever, increased virus susceptibility with blockage of phagocytes (Stairs, 1964), suggests that only those virus particles in excess of phagocytized nunbers are replicated and do so in other cells. If the dyes used to block phagocytosis did not merely occupy the cell, but actually poisoned the cell so that replication of virus did not occur, then the results of the blockage studies are compatible with the present conclusion: non- occluded particles are phagocytized and replicated in the same cell. This is not to imply that phagocytosis is necessary for the entry of virus into hemocytes for replication. The rapid ra te of increased numbers o f infected hemocytes suggests a logarithmic type of increase in nunbers of infectious particles. A cyclic theory of virus replication and reinfection provides enough part- 48 ides to infect hemocytes in a rapid, step-wise fashion. Such a cydic theory has been proposed in the past (Aizawa, 1959; Bergpld, 1958; Bird 1959). The main difference in versions of this theory was the particle

form which was thought to be responsible for reinfections. Data pre­ sented here demonstrated that once established (one infected hemocyte

in 1 ,0 0 0 hemocytes), the spread of disease was similar in all injected

larvae. Hemocyte infections have a definite relationship to the pathogenesis of NPV disease. From the onset of visual polyhedra in hemocytes, death occurs within 4-7 days. Once infection is established*in hemocytes, death seems ce rta in . Hemocytes may be more important to pathogenesis of th is virus than previously thought• By the time 20-30% of the hemocytes are infected, fat bodies were observed to be full of polyhedra. Considering that it would take fewer cycles and less time to infect the total popu­ lation of fat body cells than to infect a total population of hemocytes, the initial infection may actually be in the hemocytes. Possible role of nucleic acids.--No infectious ENA, naked or exposed,

was detected free in infected hemolymph.. The infectious ENA is apparently protected by the organization of the particles. One explanation of in­ creased infectious activity from the ENase-treated virus suspension is that the enzyme "cleaned" the activity sites or exposed new activation sites. Or, ENA might have been activated. ENA may be activated as a template-primer for certain ENA polymerases by single-stranded scissions created by pancreatic DNase (Komberg, 1974). A trace of such an endo­ nuclease activates, but an excess would lead to total destruction of ENA

a c tiv ity . 49 Another explanation for DNase increasing the NPV infectivity is that the incubated buffer-plus-enzyme lowered the resistance of larvae to virus infection. The mortality caused by injection of these chemicals without the virus supports this idea. Decreased host resistance is also reflected in the increased rate of spread of disease among hemocytes. Further demonstration of the stability of non-occluded infectious particles and their suitability as cell-to-cell agents would be the iso la tio n of a NPV-induced DNase. Herpes-induced DNase can be separated from cellular DNase by DEAE -cellulose chromatography (Morrison and Keir, 1968). Herpes serves as a model due to its weight (54-92) and s ite of replication (nucleus) being similar to NPV. The herpes-induced DNase acts extranuclearly whereas the cellular DNase acts endonuclearly. A ♦ nuclease, or similar acting material, is found in vaccinia virus parti­ cles, and may be responsible for inhibition of DNA synthesis in infected cells (Cotarella et al., 1972; Pogo and Dales, 1973). RNA appears to be an important part of infection of NPV. Either

RNase penetrated the virions to reach activity sites, or the substrate sites are outside the core. Since DNase did not penetrate to the genome, it is unlikely that RNase did. Vaccinia virus has been demonstrated to have RNA both within and outside the virion (Planterose et a l., 1962). It has-also been reported that RNA has a rule in DNA replication of human lymphocytes, E. coli phage, E. coli, and a slime mole (Fox et a l., 1973;

Lark, 1972; -Sugino e t a l ., 1972; Wager and Huberman, 1973).

Ribonucleotides covalently bonded to DNA have been detected in T 4 bacteriophage (Speyer et a l., 1972), but did not appear to be covalently linked to DNA in purified virions of vaccinia virus (Roening and Holowczak, 50 1974). NPV is similar to pox viruses in that both are large compled DNA viruses. It therefore seems reasonable to expect similarities in function

as well as structure. Vaccinia can release endogenous enzymes which can synthesize mRNA (Kates and Beeson, 1970; Kates and MacAuslan, 1967). I f such is the case with NPV, some RNA would be outside the core but beneath

the surface. The association of RNA with infectious particles in hemolymph might

occur during virus assembly or synthesized as early RNA in a new infec­ tion. The in vivo treatment of RNase could inhibit the activity of "late" RNA outside the core, but does not preclude activity of "early" mRNA in a new infection. Inclusion of RNA could be accidental and be either of

viral or of host origin. Speculation as to the possible role of RNA in­ cludes both the establishment of infection as well as virus replication. Specific sequences of RNA may be needed for infection and/or virus re­ plication. Another possibility of the role of RNA is that host-induced

RNase binds to activity sites to inhibit viral activity, thus increasing host resistance. Interference of host development.--There are two classes of death • in larvae inoculated with NPV: (1) NPV infection confirmed by visible

appearance of replicated polyhedra and ( 2) those deaths appearing to be typical of NPV infection but without polyhedra replication observed. The latter may be considered virus-associated deaths. The presence of in­ fectivity needs to be confirmed in the absence of newly replicated mature polyhedra. If replication has occurred, non-occluded infectious particles

may be present. Virus-associated deaths occurred from inoculation of alkaline-released polyhedral frozen less than 2.5 years, highly diluted 51 inocula of fresh infected hemolymph.

Mortality occurring later than 10 days post-injection resulted in either prolonged larval state or allowed the injected larvae to develop into post-larval stages. Post-larval mortality occurred in larvae inoc­ ulated with highly diluted fresh infected hemolymph, alkaline-released infectious particles from refrigerated polyhedra incubated 24 hours with NaCl, refrigerated polyhedra incubated with alkaline solution 24 hours,

1/ 2 -hours alkaline-treated frozen polyhedra supematent, and occluded particles released from polyhedra frozen 2.5 years and been incubated

20 hours with alkali. Late deaths decreased in number when test groups were injected with alkaline-treated fresh infected hemolymph compared to no-alkali treatment of infected hemolymph. Polyhedra were observed in some but not all late deaths. From these results, the late deaths with confirmed virus infection are probably the expression of late establish­ ment of virus infection in larvae injected with very low (expected levels) of infectivity. Larvae with more resistance to the establishment of in­ fection might never have mature replicated polyhedra prior to death. In such cases, the cause of death was not the destruction of hemocytes by replication and release of mature polyhedra. Delayed development has been discussed by many investigators as being the result of hormone imbalance (Reddy and-Krishnakumaran, 1974). Although some juvenile hormone may be stimulated in response to the physical injection itself, controls in the present studies indicate that the virus or possibly the ionic concentration-composition also elicit a response similar to that expected in prolonged juvenile hormone produc­ tion.or inhibition of ecdysone. 52 Subclinical levels of infection may be sufficient for a chronic in­ fection slowly leading to death. Investigations of hormonal imbalance in virus infection and in chronic infection will provide information con­ cerning resistance mechanisms of these in sects. Other variations in results may be due to physical distribution of infectious and non-infectious virus particles both in hemolymph and re­ leased from polyhedra. Filter variation does not affect results more than 10 % from filter to filter among the lOOnm pore size. Another variation is the chance position and subsequent passage of active infec­ tious particles through pores of filters. 53

CONCLUSIONS

1. Non-occluded infectious particles of NPV are free in the hemolymph of infected larvae and they can be separated from occluded particles by filtration. Since polyhedra are greater than 500nm in diameter, they are retained by the 450nm pore size filter.

2. Non-occluded virus particles are fully competent to induce infections in hemocytes, i.e., serial passage of non-occluded virus particles in infected hemolymph produced newly replicated polyhedra as well as non- occluded infectious virus.

3. The viral envelope of NPV particles does not seem to be necessary for the infection of larval hemocytes. EM study showed no enveloped particles in the infectious filtrate that had passed through a lOOnm filter. Observed rods were naked.

4. The recovery of infectious activity from stored NPV is dependent upon time, temperature, and alkaline treatment (ionic strength and dur­ ation). The quality of infectious activity decreases with time at -10°C. Freezing may alter the integrity of the polyhedral crystal. Increased ionic strength of alkali released activity in frozen polyhedra which was not released by the standard method. 54 5. Non-occluded infectious NPV particles are more resistant to treatment with alkaline solution and salt compared to alkaline- released NPV occluded particles .

6 . A cyclic process of replication and reinfection occurs during the disease process of NPV. Once infection is established in a few cells, replicated non-occluded particles infect other cells. Within 24 hours, post-injection of non-occluded particles, new polyhedra were formed in hemocytes. Subsequently, cells produced polyhedra at a rapid Clog) rate of increase. Infection is probably established by a few particles and is not based on the activity of many introduced particles. Once infection is established in larval hemocytes, death is predictable. All larvae with infected hemocytes died 4-7 days following the first appearance of poly­ hedra in hemocytes.

7. The onset of disease varied according to the concentratiohs of the virus injected. Variations in establishment of the disease at a given concentration are indicative of variable host resistance. Species resis­ tance regulates the response to concentration, and individual resistance • of host larvae regulates variations in incubation period at a given concentration.

8. Low concentrations of injected virus suspensions allowed continued development of the host to pupal and adult stages. Development processes appeared abnormal in rate of change and in structure. Arrested physiology appears to be related to delayed establishment of infection. 55 9. RNase may decrease the infectious activity of non-occluded free virus particles injected into larvae.

10. Present tests did not identify the presence of infectious naked

DNA of NPV.

11. Polyhedra were observed in 90% or more of the hemocytes. Apparently the phagocytic blood cells also support viral replication.

12. The major infectious entity is probably the rod-shaped structure which is 37nm in diameter. 56

SUMMARY

Larvae of Galleria mellonella (L.) were infected by intrahemocoelic injection of nuclear polyhedrosis virus (NPV) and maintained under breeding (colony) conditions until death. Occluded NPV particles were obtained from polyhedra in aqueous solution ( 8°C), freshly infected hemolymph, and infected hemolymph that had been frozen (-10°C) more than 12 months; non-occluded virus particles were obtained from freshly infected hemolymph. Optimal storage of infectivity was the polyhedral form of the virus in aqueous solution at 8°C. Once infection was estab­ lished in a few cells, the disease spread at a rapid rate indicating a cyclic process of replication and reinfection. Variations in establishment of infection (at a given concentration of injected virus) are indicative of a host variability. Non-occluded virus particles were sequentially passed to new larvae without the loss of virulence to the host. Virus suspensions were treated with alkaline solution, DNase, and RNase and then tested for levels of infectious activity. Virions were susceptible to RNase and alkali. The alkaline lability suggested*that RNA is present. Alkaline-released virions were more susceptible to RNase and alkaline treatment than non-occluded particles. An EM study of hemolymph showed mostly naked nucleocapsids plus other smaller particles present. Since microfiltration studies determined that the smallest infectious particle is larger than 50nm, the naked nucleocapsids are probably the agent of disease transmission from cell to cell within the host. Table 1. Classification of insect viruses according to the system of the International Committee for the Taxonomy of Viruses (Fenner, 1976) .

ICNV Names P ro to ty p e Dimensions Mol. Wt. Insect Viruses (Fam ily) V iru s (nm) (d a lto n s x 1 0 °) ENA Picornaviridae polio 20-30 2 .5 acute bee paralysis

Rhabdoviridae rabies and vesicular 175 x 70 3.5 Hart Park virus, stom atitis virus sigm a v .

allied to Reo- 60 1 2 .7 Cytoplasmic Poly. v ir id a e R e o v irid a e r e o v iru s 75-80 15

DNA Parvoviridae Latent Rat virus 18-22 1 . 2- 1.8 densonucleosis G. mellonella Baculoviridae NPV of Bombyx mori 40-70 x 250-400 80 NPV and Granulosis Iridoviridae Tipula iridescent v. 130 130 SIV, TIV, CIV

Poxviridae vaccinia 170-250 x 300-325 160 entomopoxv. tn ^ 3 Table 2. Distribution of insect viruses within the format of the system by Lwoff, Horne, and Tournier (Lwoff et al., 1962).

Nucleic Capsid Acid Symmetry E nvelope Insect Viruses Group Prototype

DNA H e lic a l P r e s e n t' NPV (Borrelina, B irdiavirus); GV (Bergoldia Pox virus virus); poxviruses (vagiovirus) A b sen t None s-stranded phages ( f d . M13) Cuboidal Present None Herpes, Adeno­ virus , Phages Papillovirus Absent Refringent types (Paillotella); denso- nucleosis virus of Galleria (Kurstak and Cote, 1969)

RNA Helical Present Sigma virus of Drosophila Myxovirus, P a ra m y x o v iru s, Rhabdovirus, Leukovirus Absent None rod-shaped plant v i r u s e s . Cuboidal Present None Encephalovirus A bsent CPV (Borrelina, Sm ithiavirus); bee Picom aviruses, p a r a l y s i s d-stranded RNA

in 00 Table 3. An example of synonyms for insect viruses, e. g., the nuclear polyhedrosis v ir u s (common nam e).

...... NAMES...... REFERENCES

Nupoviridae, family for NPV alone Harrap and Tinsley, 1971 Baculoviridae, family for NPV F e n n e r, 1976 (ICTV) ■ and GV Baculovirus, genus for NPV Baculovirus, genus for NPV and GV W ild y , 1971 (ICNV)

N u c le a r p o ly h e d r o s is , common name Bergold, 1947 based on site of replication and form

B orrelinavirus, NPV and CPV B e rg o ld e t a l . , 1960 (ICNV) Birdiavirus, NPV in sawflies B erg o ld e t a l . , 1960 (ICNV)

m 60

Table 4. Percent of nucleic acid base content (G + C) and particle weight of groups of DNA viruses (Wildy, 1971) .

ICNV G + C Particle Weight

Nomenclature Content (daltons x 1 0 ®)

Parvovirus 39% 1 . 2- 1 . 8 L ip o v iru s Polyoma virus 41-49% 3.0 Papilloma virus 49% 3 .0 Caulimovirus 5.0

A denovirus 48-57% 20-25 Caudaevirus 50% 32 Herpesvirus 57-74% 54-92

Baculovirus 35-59% 80 Iridovirus 29-32% 130

M yovirus 34% f1) 130

P o x v iru s 35-40% 160-200 (1) Cytosine replaced by 5-hydroxymethycytosine. 61

Table 5. Nucleic acid base composition of RNA in polyhedra protein compared to bases of host silkworm RNA.

Nitrogen Base NPV polyhedra*3 >c H osta '*b guanine 16.6 29 adenine 36.8 24

c y to s in e 8 .1 25

uracil 38.9 22

a reference: Eto, 1955

^Expressed as moles percent of total bases cAfter perchloric acid treatment 62

T a b le 6 . Effect of storage of infected hemolymph in larvae a t -1 0 °C.

Infected Larval I n f e c te d T o ta l Period Frozen Parts per ml. L a rv ae Deaths Adults

32 m onths 2 x 1 0 " 1 0 /3 0 0/30 29/30

30 m onths 8 x 1 0 _1 0 /1 5 0/15 15/15

12 m onths 4 x 10 "1 0 /1 5 0/15 15/15

1 1 /4 m onths 2 x 1 0 _1 3 8 /3 8 38/38 none

4 m onths 2 x 1 0 _1 1 0 /1 0 1 0 /1 0 none 63

Table 7. Effect of storage on infected hemolymph in live larvae and cadavers at -10°C. Occluded virus part­ icles were released by incubation in alkaline solu­ tio n 1/ 2- h r .

Condition of Time Infected Larval I n f e c te d T o ta l Stored Larvae F ro zen Parts per ml. L arv ae Deaths Adults

Dead 32 m os. 2 x 10 _1 0/30 9/30 21/30

A liv e 30 m os. 4 x 10 " 1 12/14 15/15 none

Dead 28 mos. 2 x 10 _1 0/30 4/30 25/30

A liv e 12 m os. 4 x 10 " 1 1/15 2/15 13/15 64

T a b le 8. Effect of various ionic concentrations of Na 2C0 _- NaCl on infectious activity of released particles from refrigerated (8°C) polyhedra in aqueous solution.

Ionic Percent Concentration (Na?COy - NaCl) Infectivity

0 . 11M 28% 0.10M 19% 0.05M 100% 0.0 3M none Table 9. Effects of storage (-10°C), alkaline ionic concentration and duration of alkaline solution (pH 7.5) incubation on infectious activity of infected hemolymph.

Duration Ionic Con- D ise a se d Percent In­ T o ta l of Storage centration (Na^COq-NaCl) L arv ae fected Larvae Deaths Adults 30-minute Incubation:

12 mos. 0 . 1 0 M 1/15 7% 2/15 13/15 30 m os. 0 . 10M 12/14 92% 14/14 none 30 mos. 0 .05M 0/30 none 5/30 25/30 34 mos. 0.05M 0/30 none 9/30* 21/30

20 -hour Incubation:

12 m os. 0 . 10 M 9 /1 3 69% 9 /1 3 4 /1 3 30 mos. 0 . 10M 2 /1 2 17% 2 /1 2 1 0 /1 2 30 mos. 0.05M 0/30 none 5/28 23/28 34 m os. 0 .05M 0/30 none 25/30* 5/30

*Larvae from A. Pye's s t r a i n " 0 "

cn 66

Table 10. Effect of storage period on infectivity released from polyhedra in aqueous solution at 8°C .

Virus Percent Storage Period Concentration Infected

1 y r . 5 mos. 4 X 10 7 p o ly /m l 8 8

1 y r . 7 mos. 4 X 10 7 p o ly /m l 100

2 yrs. 3 mos. 4 X 10 7 p o ly /m l 100

2 y r s . 4 mos. 4 X 10 7 p o ly /m l 30 (D

5 y r s . 1 mo.* 2 . 6 X 10® p o ly /m l 76

5 y r s . 2 mos. * 3.0 X 1 0 6 p o ly /m l 60

5 y r s . 5 mos. * 4.0 X 10 3 p o ly /m l 40

♦clum ping

^L ast instar larvae injected Table 11. Effect of 20-24 hours NaCl (0.045M) on infectious activity of refrigerated polyhedra (8°C) and of fresh infected hemolymph. Na^CC^ (0.0025M) was added to refrigerated polyhedra 1/2 hour prior to injection.

V iru s o n ly______NaCl NaCl+Na-jCO-a______

Larvae Surviving Larvae Surviving Larvae Surviving Infected Adults Infected Adults Infected Adults

Fresh Infected 12/12 1/15 1 5/15 none 15 /15 none hemolymph

Refrigerated ------0 /6 1/14 7/1 2 2/15 Table 12. Effect of serial passage, filtration, and dilution on infectivity of in­ fected hemolymph from diseased live larvae.

C onfirm ed Passage Concentration No. la r v a e V iru s T o ta l Nov Infected Hemolymph T re a tm e n t i n j e e t e d i n f e c t i o n M o r ta lity (Infected larval p a r ts /m l)

1 p o ly h e d ra . A lk a lin e + 450nm 50 10/35 43/50 2 2 .0 X 450 + 220nm 26 26 26 10 1 3 1 .3 X 1 0 ” 1 45% sucrose grad. 15 15 15 + 450nm 4 1 .8 X 1 0 -2 22 0nm 50 50 50 5 1 .8 X 1 0 -2 450 + 220nm 20 20 20 6 1 .8 X 1 0 " 2 450 + 220 +100nm 20 4 4 7 450;dil. 1/3; 220nm. 20 20 20 8 2 .0 X i o - i 450 + 220 + 100(10~2) 10 10 10 9 2 .0 X 450 + 220 + 100(10"3) 10 2 3 10 1 10 2 .0 X 450 + 220nm (10-3l 10 10 10 10 l 11 2 .0 X 450 + 220 nm(10 ) 10 10 10 1 0 - 1 12 2 .0 X 10 1 450; refrig. 9 days; 5 5 5 450nm repeated 13 2 .0 X io “ i 450 + 2 2 0 (1 0 -°) 10 4 7 14 2 .0 X 1 0 " 1 450nm 10 10 10

oo 69

Table 13. Calculated slopes (b)r intercepts, and LDso's °f filtratio n experiments (Data in Appendix A and graphed in Figure 1, A-F.).

F i l t e r S lope Log E xperim ent (nm) (b) I n t e r c e p t LDc;n

A. 450 0.52 5.6 - 1 . 2 220 0.36 4.3 +1.7

B. 220 1.70 9 .7 - 2 .7 50 0.36 4.0 + 2 .7

C. 220 1.28 8.5 - 2 . 8 1 0 0

D. 220 1 0 0 1.15 9.9 -4.2

E. 10 0 0.61 7 .7 - 4 .4 Table 14. Polyhedron formation in hemocytes and infection of larvae.

Days Post- Percent Hemo- No. of Percent larvae E x p erim en t Injection cytes Infected Larvae Infected

A. 1 0 .5 (0 . 1 - 1 , 0 ) 25 83 2 1 2 .0 (5-30) 25 100 3 6 0 .0 (20-90) 25

6 A ll Dead

B. 1 0 5 0 2 1 2 .0 (5-30) 5 100 3 60.0 (20-90) 5

6 A ll Dead

1 0 .3 (0 . 1 - 0 .5) 20 12 2 6 .0 (0 . 1- 2 0 ) 20 53 3 35.0 (20-50) 34 89 4 77.0(50-90) 38 100

6 A ll Dead 71

Table 15. Effect of DNase on infectivity of non-occluded particles when incubated for 20 hours.

Virus Dilution (Inf. Larval Parts Infected Infected hemolymph per ml) ' Larvae Adults

No DNase 1 0 - 7 3/10 7/10 1 0 -1 0 5/10 5/10

lOOug/ml DNase 1 0 " 7 1 0 /1 0 0 /1 0

1 0 - 1 0 9/10 1 /1 0

200ug/ml DNase 10“ 7 9/10 1 /1 0

lO'-lO 9/10 1 /1 0

Tmzyme w ith buffer

lOOug (50% post-larval deaths) 0 /1 0

2 0 0 ug (All deaths post-larval) 8 /1 0

No treatment 0/10 7/10 72

Table 16. Effects of DNase on the infectivity of alkaline 1 released particles.

A lk a lin e Dead DNase Solution Buffer

Virus diseased 0/29 23/30 0 /2 0

Other 5/29 7/30 4 /2 0 Surviving Adults 24/29 0 /3 0 16/20 Table 17. Effect of ribonuclease on infectivity of infected herolyrnph (06.5 x 10 “2 infected larval parts per ml) * injected into healthy larvae (Experiment #41, Appendix A).

Fraction Percent Dead Incubation Fraction of Survival to w ith :(24°c) Needle Deaths as larvae' as Prepupae' as Pupae' ' Adult Stage Polyhedra VIRUS

Alone 20 hours 1/10 9/10 0 0 0 100% 100 ug RNase 20 hours 2/10 8/10 0 0 0 100%

200 ug RNase 20 hours 0 10/10 0 0 0 100%

ENZYME CONTROLS

lOOug RNase 20 hours 0 6/10 2/10 1/10 1/10 0 200ug RNase 20 hours 0 4/10 4/10 0 2/10 0 100 to 200ug 0.5 hours 0 0 0 0 10/10 0 RNase

' V ...... V S \ N. •. •, v v v V \ V

''J 74

Table 18. Dilution levels of infected hemolymph producing infected hemocytes in 50% of larvae injected. Infected hemolymph was incubated 20hr with lOOug and 2 0 0 ug ribonuclease.

Dose Levels at Which 50% Larvae Became Infected No Enzyme lOOug/ml 200ug/ml - 4 " 5 v-3 Infected hemolymph 1 0 " 9t o l 0 " 12 10 tolO 10

Infected hemolymph 1 0 _ 8t o l 0 “ 9 10-3 plus alkali Table 19. Effect of alkaline treatment and filtration on infectious activity of in­ fected hemolymph. Final total concentration of NaCl plus Na 2 C0 , was 0.05M, pH7.5. Each virus suspension passed through a 450nm pore-size f i l t e r .

Sequence of Virus Dil. I n f e c te d T o ta l T re atm en ts (Infected larval L arv ae Deaths Adults p a r ts /m l) -8 Alkaline solution—filtered 7 10 8/ 8* 8 /1 0 2 /1 0 -11 7 10 1 /1 0 5 /10 5 /10 -3 Filtered—alkaline solution 7 x 10 1 0 /1 0 1 0 /1 0 none 7 xx 1 0 “ 5 8 / 8 8 /8 none 7 x 10 -8 6 /7 * 7/9 1/9 7 x 10 -11 3/5* 5 /10 5 /10

Filtered only 13 x 10 " 8 9 /9 9 /9 none 13 x 1 0 -H 8/ 8* 8 /1 0 2 /1 0 *Pupal hemolymph not examined to avoid injury. Figure 1. Comparison of the infectious activity of virus particles that pass through 450nm and 220nm filters with mortality plotted in probits over a range of log doses . Observed probits (X ) have been used to calculate probits Co) according to Bliss (1938). The slopes are sim ilar; LD50 with 450nm filter is approximate­ ly 10~2 log doses, LD50 with 220nm filte r is approximately 102 log doses. 76 9 4 5 0 nm filter 8

oL. CL

-6 •4 •2 0 log Dose

220 nm filter

log Dose Fig. 1 Figure 2. Comparison of th e infectious a c tiv ity of virus p artic le s th a t pass through 220nm and 50nm filters with mortality plotted in probits over a range of log doses. Observed probits (X) have been used to calculate probits (o) according to Bliss C1938). The difference in activity rates is discussed in the test. The LD50 with 220nm filte r is approximately 10"2 .5 log doses; LDtjo with the 50nm filte r is approximately 1(A log doses. This data does not reflect replication of polyhedra. 77 220 nm filter

M 15 o Q.w

log Dose

9 50 nm filter 8

7

6

(A c 15 5 2 CL 4

3

2

1

log Dose Fig. 2 Figure 3. Comparison of the infectious activity of virus particles that pass through 220nm and lOQnm filters with mortality plotted in probits over a range of log doses. Observed probits QO have been used to calculate probits (o) according to Bliss (1938). The similarity of the LD50 values is discussed in the text. The difference in rates is attributable to the range of results as discussed in the text. 78 X 9 220 nm filter

•QO

■6 ■4 2 log Dose

100 nm filter

■OO

■4 -2 0 log Dose Fig. 3 Figure 4. The infectious activity of virus particles that pass through lOOnm filte r with mortality plotted in probits over a range of log doses. The activity points of virus particles that passed through the 220nm filte r in the same experiment has similar plot points; see text for discussion. Observed probits (X) have been used to calculate probits Co) according to Bliss (1938). The LD50 i s 10“4 log doses. 79

100 nm filter

-6 ■4 2 0 log Dose Fig. 4 Figure 5. The infectious activity of virus particles that pass through lOOnm filte r with mortality plotted in probits over a range of log doses. Observed probits (X) have been used to calcu­ late probits Co) according to Bliss (1938). The LD50 is 10"4 log doses.

> 80

100 nm filter

o Af>

1 -4 0 log Dose Fig. 5 \

Figure 6 and 7. An electron micrograph (280,000- ■%)■ of -highly infectious (MV) body fluids of a larva of' Galleria tnelldriella. (2a.) and (3a) are virions (36 nm in diameter) that had passed through a lOOnm microfilter. Both are bent and broken (disrupted) in several locations. (2b) and (3b) are particles 15-20 nm in diameter. (2c) and (3c) are p a rtic le s 7-9 nm in diameter. 81 82

DNase ciOQug) £ o a) bo rj rj U rt rd O u ssO R)a

PERCENT INFECTED CELLS PER LARVA

CONTROL

0) o q) 60 £ 2 Rj R) iJ O O• ^3O Z

PERCENT INFECTED CELLS PER LARVA

Figure 8, Percent of hemocytes with polyhedra per 100 hemocytes two days post-injection with virus suspension. APPENDIX A EFFECT OF FILTER SIZE ON INFECTIVITY OF INFECTED HEMOLYMPH. i

L arv ae I n ­ Percent M ortality Probit M ortality Exp. Filter V iru s D il. Total fected with NO. (nm) (lo g ) Larvae Polyhedra Larvae! Total Larvae T o ta l

A. 450 - 2 10 2 / 1 0 20 30 4 .1 5 4 . 5C -3 10 2 / 1 0 20 20 4 .1 5 4 *°c -4 10 none none 10 2 .6 7 3 *5c -5 10 none none none —----- 3.0

2 2 0 - 1 10 1 / 1 0 1 0 10 3 .7 1 4 *°c - 2 10 none 10 2 0 * 3 .7 1 3 .6 ° -3 10 none none none** 2 .6 7 3 .4

B. 220 -1 10 10/10 100 1 0 0 8.09 8. 0 ° - 2 10 1 0 /1 0 90 1 0 0 6 .2 8 6 .3 C -3 10 none 10 10 3 .7 1 4 .6 -4 10 none 10 10 3.71 2 . 8°

50 - 0 10 none 10 10 3.71 4 *1c - 1 10 none 10 20 3 .7 1 3 .7 ° - 2 10 none none none 2.67 3.4° ♦Injured pupae died. ’'^uannaoaiism. oo W CONTINUATION OF FILTER EXPERIMENTS(pg.2)

Larvae In- Percent Mortality Probit Mortality Exp. Filter Virus Dil. Total fected with No. (nm) (log) Larvae Polyhedra Larvae Total L arv ae T o ta l

C. 220 - 2 10 1 0 / 1 0 1 0 0 1 0 0 8 .0 9 7 .2 ° -3 10 8 / 1 0 50 80 5 .0 0 5 .9 -4 10 1 / 1 0 30 40 4 .4 7 4 .7 °

1 0 0 - 2 10 none none none ______-3 10 2 / 1 0 30 60 4 .4 7 5 .2 5 -4 10 none none 40 2 .6 7 4.74

D. 220 -1 10 10/10 1 0 0 1 0 0 - 2 10 1 0 / 1 0 1 0 0 1 0 0 8.09 -3 10 1 0 / 1 0 50 80 5 .0 0 -4 10 1 0 / 1 0 1 0 0 * 1 0 0 * ------

100 -1 10 10/10 1 0 0 1 0 0 ------8 . 8° - 2 10 10/10 100 100 8.09 7 *6o -3 10 4/10 50 90 5 .0 0 6'3c -4 10 2 / 1 0 30 60 4 .4 7 5 .2 ° ♦Small, young larvae. CONTINUATION OP FILTER EXPERIMENTS (pg. 3 ).

Exp. F i l t e r V iru s D il. T o ta l fected with NO. (nm) (lo g ) Larvae POlyhedra Larvae Total Larvae T o ta l

E. 450 -5 10 1 0 / 1 0 1 0 0 1 0 0 M M M -7 10 9/10 1 0 0 1 0 0 ------

2 2 0 -7 10 1 0 / 1 0 * 1 0 0 1 0 0 ------

1 0 0 - 1 10 1 0 / 1 0 1 0 0 1 0 0 8.09 7 .0 ° -3 10 9/10 62.5 87.5 5 .3 3 5 . 8C -5 10 1 / 1 0 10 20 3 .7 1 4 .6 C -7 10 none none none 2 .6 7 3. 8C

P . 450 -9 1 0 1 0 / 1 0 1 0 0 1 0 0 ------

2 2 0 - 0 10 10/10 100 100 8.09 -3 10 10/10 100 100 ------6 1 0 4/10 40 40 ——

1 0 0 - 0 10 9/10 1 0 0 1 0 0 8.09 -3 10 none none none 2 .6 7 - 6 10 none none none ------

* cProbits calculated according to method for small numbers (Bliss, 1938). Experiment. #41. Infectivity of alkaline and non-alkaline tgeated infected hemolymph incubated with ribonuclease (20 hrs. at 20 C).

No Enzyme 10 Oug/ml 2 0 Oug/ml Log % I n - % I r i - % I n - D il. D eaths ■fee te d Adults Deaths fected Adults Deaths fected

Infected Hemolymph 1010"1 1 0 /1 0 100 0 1 0 /1 0 100 0 1 0 /1 0 100 0 (no alkali) i o ” 3 1 0 /1 0 100 0 * 7 /1 0 70 3 1 0 /1 0 62 0

io-6 10/10 100 0 6 /1 0 40 4 ------9 10 8 /1 0 80 2 8 /1 0 none 2 ______

38 /4 0 2 3 1 /4 0 9 2 0 /2 0

IO"1 1 0 /1 0 100 0 1 0 /1 0 100 0 ------A lk a lin e T re a te d io " 3 7 /9 88 0 9 /1 0 50 1 ------

io " 6 6 /9 77 1 ------— 4 /1 0 40 6

10"9 6 /1 0 60 5 ----- — 1 /1 0 none _9_

2 9 /3 8 6 19 /2 0 1 5 /2 0 15

00ON 87 APPENDIX B ESTIMATION OF LD - VALUES FOR SMALL NUMBERS ACCORDING TO METHOD OF BLISS (1938)

The expected dosage-mortality curve is computed by least-squares from the logarithims of the doses (x values) and the observed probits (y values) . The squares and the

products of the deviations are totalled to determine the regression coefficient.

B= estimate of b= E(x-x) (y-y) (D E (x-x) New probit values (y) are calculated from the expected regression line.

y= y + B(x-x) (2 ) This dosage-mortality curve is w ritten as Y = a + bX (3) From this provisional line, the probits are corrected and weighted as recommended for small numbers, e.g., 10 p e r test group (Bliss). Correction of probits uses a weighted 2 coefficient (z_) giving more weight to probit values between pq 4 and 6 than to other values. As stated by Bliss, z is the ordinate of the normal curve corresponding to the observed

proportions of dead (p) and living (q = 1 -p) host individuals in a given sample. Corrected probits (y) are computed from

expected (provisional) probits (Y) y = Y + Q -

^w served probit values. Weighted probits (Pw) are calculated using equation No. 2. 89

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