Group Title: cytopathology of a nuclear polyhedrosis virus in Aedes triseriatus (SAY)
Title: The Cytopathology of a nuclear polyhedrosis virus in Aedes triseriatus (SAY)
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Permanent Link: http://ufdc.ufl.edu/UF00098208/00001
 Material Information
Title: The Cytopathology of a nuclear polyhedrosis virus in Aedes triseriatus (SAY)
Physical Description: 98 leaves : ill. ; 28 cm.
Language: English
Creator: Federici, Brian A., 1943-
Publication Date: 1970
Copyright Date: 1970
 Subjects
Subject: Viruses   ( lcsh )
Virus diseases   ( lcsh )
Insects -- Diseases   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Brian Anthony Federici.
Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Bibliography: leaves 92-97.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00098208
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000425450
notis - ACH3971
oclc - 81547338

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THE CYTOPATHOLOGY OF A NUCLEAR POLYHEDROSIS

VIRUS IN Aedes triseriatus (SAY)














By
BRIAN ANTHONY FEDERICI












A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY















UNIVERSITY OF FLORIDA
1970













TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS .................................. ii

LIST OF TABLES .................................... v

LIST OF FIGURES ....... ... ....................... vi

ABSTRACT .......................................... xii

INTRODUCTION ...... ............................. 1

LITERATURE REVIEW ........... ................. ... 5

MATERIALS AND METHODS ............................ 16

Larval Rearing and Colony
Maintenance ................................. 16
Inoculation of Larvae ......................... 17
Mortality Studies ............. ..... .. ........ 18
Transovarial Transmission
Studies ............................... .... 18
Histology and Cytopathology ................... 19
Measurement of Viral Components ............... 21
Chemical Behavior of Polyhedra ................ 21

RESULTS .......................................... 23

Pathology .................................... .. 23
Cytopathology .................................. 36
Mortality Studies ........ ..................... 61
Transovarial Studies .......................... 63
Chemical Behavior of Inclusion
Bodies .................................... .. 63

DISCUSSION ........................................ 66

Appendix

1. Staining Procedures for Light
Microscopy ................ ............... 80










TABLE OF CONTENTS-Continued


Page

Appendix

2. Fixation and Embedding Schedule
for Electron Microscopy ................. 83

3. On the Terminology Applied to the
Morphology and Anatomy of
Nuclear Polyhedrosis and
Granulosis Viruses ...................... 84

BIBLIOGRAPHY ................................... .. 92

BIOGRAPHICAL SKETCH ............................. 98













LIST OF FIGURES


Figure Page

1. Section Through the Stomach of a
Third-instar Control Larva Stained
with Hematoxylin and Eosin .............. 27

2. Section Through the Infected Stomach
of a Third-instar Larva Stained
with Hematoxylin and Eosin .............. 27

3. Section Through Infected Cardia Cells
of a Third-instar Larva Stained
with a Hamm's Stain ..................... 27

4.- Section Through a Heavily Infected
Gastric Caecum in a Third-instar
Larva Stained with Hamm's Stain ......... 27

5. Section Through Cells in the Posterior
Part of the Cardia, Showing the
Infected Cells in This Area Stained
with Hematoxylin and Eosin .............. 30

6. Section Through an Area Where There
Appeared to be a Proliferation of
Infected Cells ............................ 30

7. Section Through an Infected Nucleus,
Showing a Developing Virogenic Stroma
Stained with Hematoxylin and Eosin ...... 30

8. Section Through Infected Stomach Cells,
Illustrating Different Stages of
Polyformation ...................... ...... 30

9. An Oblique Section Through the Midgut of
an Infected Third-instar Larva .......... 33

10. An Oblique Section Through the Infected
Midgut of a Third-instar Larva .......... 33

11. A Polyhedra Dissociating in the Midgut
Lumen .................................... 35










LIST OF FIGURES--Continued


Figure Page

12. A Polyhedra Dissociating Between the
Peritrophic Membrane and the
Microvilli .......... ... ............. 35

13. Virions Accumulated Along the
Peritrophic Membrane at the
Site of its Formation ................ 35

14. Polyhedra and Free Virus in the Area
Between the Microvilli and the
Peritrophic Membrane in the
Stomach .............................. 35

15. Virions in an Area Between the
Peritrophic Membrane and the
Microvilli of the Stomach ........... 35

16. Two Virions "Attached" to Microvilli
in the Stomach ....................... 35

17. A Healthy Midgut Epithelium Cell
From the Stomach of a Third-instar
Larva ............................... .. 39

18. An Infected Nucleus in which the
Nucleolus has Moved to the Edge
of the Nuclear Envelope and Begun
Dividing .............................. 39

19. A Nucleus in an Early Stage of
Infection with the Virogenic Stroma
Easily Visible in the Central Area ... 39

20. The "Cords" of a Well-developed
Virogenic Stroma ..................... 39

21. A Helical Coil Typical of Those
Occasionally Associated with the
Virogenic Stroma ..................... 39

22. Capsids in an Early Stage of
Formation ............ ...... ..... ..... 39

23. Cross and LOngitudinal Sections
Through Two Regular Arrays of
Capsids ............................. .. 42










LIST OF FIGURES--Continued


Figure Page

24. A Cross-section Through an Aggregation
of Capsids ............................... 42

25. A Cross-section Through an Aggregation
of Newly Formed Nucleocapsids ........... 42

26. An Aggregation of Newly Formed
Nucleocapsids ...................... ..... 42

27. A Cross-section Through a Single
Nucleocapsid ............................. 42

28. A Freeze-etch Replica of an Infected
Nucleus .................................. 42

29. A Section Along the Longitudinal Axis
of a Nucleocapsid Aggregation ........... 46

30. A Section Along the Longitudinal Axis
of a Nucleocapsid Aggregation in
which the Shorter Nucleocapsids Give
the Impression of Having "Budded" Off
the Larger Nucleocapsids in a
Longitudinal Direction .................. 46

31. An Infected Nucleus Cut at a Plane in
which the Majority of Nucleocapsids
are in Parallel Aggregations ............ 46

32. An Atypical Long and Curved
Nucleocapsid ............................. 46

33. Vesicular Material from which the Outer
and Intimate Membranes are
Derived .................................. 46

34. A Single Nucleocapsid Attached at One
End to a Small Vesicle .................. 46

35. Attachment and Envelopment
Nucleocapsids ............................ 49

36. Envelopment of Nucleocapsids and
Formation of the Intimate Membrane
in Small Vesicles ........... ............ 49


viii










LIST OF FIGURES-Continued


Figure Page

37. Envelopment of Nucleocapsids and
Formation of the Intimate Membrane
in Large Vesicles ..................... 49

38. Longitudinal Nucleocapsid Envelopment
in which the Unit Membrane-like
Structure to the Outer Membrane
is Apparent .......................... 49

39. Vesicular Envelopment of Nucleocapsids
in Large Vesicles ................... 49

40. An Area Where the Majority of the
Nucleocapsids have been Enveloped
Individually ........ ................. 49

41. A Section Through an Infected Nucleus
Illustrating Complete Virus Rods
Before Occlusion ..................... 52

42. A Freeze-etch Replica of an Infected
Nucleus ............................... 52

43. Freeze-etch Replica of a Developing
Polyhedra ............................. 52

44. A Freeze-etch Replica of a Developing
Inclusion ............................. 52

45. An Ultra-thin Section Through a
Developing Polyhedra ................. 52

46. A Scanning Electron Micrograph of a
Stick-like Inclusion Typical of
Those Seen in Early Stages of
Polyhedral Formation ................. 55

47. A Group of Small Stick-like and Club-like
Inclusions which have begun to
Coalesce .............................. 55

48. A Bundle-shaped Inclusion Formed by the
Condensation of Several Stick-like
and Club-like Inclusions ............. 55










LIST OF FIGURES--Continued


Figure Page

49. An Ultra-thin Section in which Polyhedra
have begun to Condense in the "Ring
Zone" of the Nucleus .................... 55

50. A Nucleus in a Very Late Stage of
Infection ................................ 55

51. A Section Through a Developing
Polyhedra in a Late Stage of
Formation ................................ 55

52. The Polyhedral Contents of a Nucleus
which was Osmotically Shocked During
an Advanced Stage of Inclusion
Formation ................................ 58

53. An Advanced Stage of Inclusion Formation
in an Infected Stomach Cell
Nucleus .................................. 58

54. A Portion of Condensing Inclusion .......... 58

55. Ellipsoidal Inclusions at an Advanced
Stage of Formation ...................... 58

56. A Stomach Cell Nucleus in a Very Late
Stage of Infection ...................... 58

57. An Advanced Inclusion in the Nucleus of a
Cell in the Gastric Caecae .............. 58

58. A Spindle-shaped Inclusion with a Smooth
Surface .................................. 60

59. Membraneous Lumellar Organelles in the
Cytoplasm of an Infected Gastric Caecum
Cell ................................... .. 60

60. A Segment of the Nuclear Membrane from
an Infected Gastric Caecum Cell ......... 60

61. An Infected Stomach Cell in which
Proteinaceous Granules have Ac-
cumulated in the Cytoplasm in an
Area of Heavy Ribosome Con-
centrations ............. ................ 60










LIST OF FIGURES-Continued


Figure Page

62. A Freeze-etch Replica of a Fractured
Surface Through a Membraneous
Organelle of the Type Found in the
Cytoplasm of Infected Cells ........... 60

63. A Membraneous Organelle Similar to the
One Above .............................. 60

64. A Schematic Illustration of Completely
Developed Non-occluded and
Occluded Virions ...................... 76









Abstract of Dissertatiion Prresnted to the Graduate Counc;l
of the University of Florrida in Partial Fulfillment of Lie
Requireiexnl.f for the Degr.ce of Doctor of Philosophy


THE CYTOPAT'HOLOGY OF A NUCLEAR POI,YI!'DR:OSiS
VilUrJ 11 AI;ED TR; ISRIATUS (SAY)


By

Brian Anthony Federici

December, 1970


Chairinma:i F. S. Blanton
Co-Chairman: R. E. Lowe
Major Departmient: Entomology'


An investigation was undertaken to dete:r mi;: t:h

histopathology, cytopathology, and moipholo)cy of a n.i.cl>r

polyh-'dW-osiJs virus (NPV)W irU Accdol t'.i re:i siu.: (S,. ay). Th'c

virus was found to attack: the card ast-ric caerna, and

stomach of the miidyut epithelium, and in mest con.es -:sY'Yied

in the death of the afflic:td larvae. The dise' .e wa:

ma:rkced by sCuggishness loss of appeti te, and the hl'er--

trop:hy of infected tissues. The moi-Lality rates were 36.5

per cent amjn 3i. 4 par cent P-es).OectJ vly, fr .- rvac ii'occ.-

3aite' i;-r 21 an id 48 hours of ago. Late third-, and ifou tii-

in:tna-r s;ho; :-dl 1 .ti c su.-cept.i.b.jity to infection.

Th'i projLOs-s. Lor f ti, di 3;.seaso v.ihin the L-.I mcl-i

.s iypic,'l o;- tha' :: .L d fo:r okb:"r NPV;., ji.'r d.i

hyir,'. ,r':. y c :1;,.r-.ct';d r cle-, iegq .ncr 1.i (:( '.f hi-i .











chromi(tin with the subscqucnt formation of a feul (en-

positive virogenic stoma, and thie eovoiit.al deve]opmen I. of

large virus occluding protein inclusions throughout the

nucleus. The rod-shaped virions measured 63 y 200 mu and

were occunded in large fusiform inclusion bodies v.hicl

measured 3-7 u in diameter by 6-20 u in leng-th.

Examinations of ultra-thin sections of infected

tissue revealed that the virus was composed of a nucloo-

capsid enveloped within an intimate membrane, of uni noivn

composition, and an outer envelope, which had a unit rmerbrane-

like structure.

Fully developed virions underwent a reoduction -in siz

during the occlusion process. Neither virio!.:o or the

structure of the crystalline latLice were ever resolved in

mature inclusions.

The results of transovarial studies were negative

although there was some evidence for trans-ovum traninmin-sion.


x i ; i.













INTRODUCTION


The control of pest species of insects, long one of

mankind's goals, is directly relevant to many of the world's

health and welfare problems such as food production, pollu-

tion, and the population explosion. Most early methods of

control were mechanical, but near the end of the 19th

century man began to employ chemical and biological methods

to aid in his quest to manage insect pest populations. The

chemical methods consisted mainly of petroleum sprays, botan-

icals, and inorganic insecticides such as the arsenicals.

Petroleum was utilized primarily for mosquito control and

the latter two types were used to control crop pests.

Early methods of biological control considered only the

introduction of predators and parasites from other geograph-

ical areas. Techniques for employing these methods were

expanded upon during the first few decades of this century.

The employment of these techniques required a detailed

knowledge of the biology and ecology of both the pest

species and control species, and there were few economic

successes.

The insect pest control situation changed dramatically

in the 1940s with the advent of DDT and the synthesis of

other chlorinated hydrocarbons. Many researchers involved








in insect control, especially those in government and in-

dustry, interested in military personnel protection, di--

rected their attention to finding other organic insecticides.

Because of this concerted effort, numerous candidate insec-

ticides were investigated and within a short time the

organophosphates and carbamates were in widespread use.

One of the direct consequences of tIhis shift to organic

insecticides was a diminution of interest in other control

methods, such as biological control, and, thus, the develop-

ment of an ecological approach to pest control fell by the

wayside.

By 1960 the widespread use of organic insecticides had

resulted in remarkable increases in food production, con-

trol cf such dread diseases as malaria and typhus, and, in

turn, in increased population growth rates. However, by the

same tine, it had become apparent that several species of

insects had developed resistance to organic insecticides,

and that certain members of the more persistent chlorinated

hydrocarbons were being concentrated at the tops of many

food chains by a process known as "biological magnification."

Although the total consequences of these processes are not

ye': known, it is becoming increasingly apparent that they

may be detrimental to the biosphere.

The prospects of increased environmental pollution and

additional resistance of pest species brought about by the

continued use of organic insecticides have once again

aroused interest in non-insecticidal methods of control.








These areas are still largely unexplored, but it is evident

that utilizing several methods simultaneously will be in-

creasingly relied upon in the future.

Mosquitoes, because of their great economic, medical,

and veterinary importance, have been the object of numerous

diversified control programs. Presently, these world-wide

programs rely heavily on the use of chemical insecticides.

Although some outstanding preliminary research has been

done in the areas of sexual sterilization and the genetic

manipulation of mosquito populations, until recently little

has been done in the area of microbial control. This is

due mostly to the fact that few effective mosquito patho-

gens were known. However, efforts to find new pathogens

have been intensified and now several pathogens have been

found which show promise as biological control agents.

In considering pathogens as potential control agents,

the insect viruses possess several properties which make

-them particularly desirable. In most cases they attack the

larval stages of the insect, they are highly virulent, most

of them are rather host specific, and to date they have

been shown to be non-pathogenic to man and other animals.

However, little is known about their structure, multipli-

cation, and biology, and it has been difficult to quantify

virus particles for dosage-mortality studies. Presently,

the main problem is the production of viruses in large

enough quantities to provide material for study and testing.








This difficulty nay be overcomne an insect tissue culture

technology is advanced.

One oF the m-nst promising of the new imo.gqMiito viruses

is the nuclear-polyhcdrosis reported from Acdes so] licitcan

(Ul7k.) by Clark et al. (1969). This virus causes rapid

infection in early, instar mosquito larvae and has been

shown to caLsrc relatively high mortalities. ChaipIan (ler-

sonal comiruniication) has transmitted this virus to several

other species of mosquitoes, including A'des triscriatus

(Say), Acdes tornientor (Dyar and Knab), Pcaorophora varipes

(Coquillett), and Psorophora ferox (iHumboldt).

The chief objectives of this study were to investigate

some aspects of the structure, multiplication, and pathology

of this nuclear--polyhedrosis in A. tri ser.iatuc. This partic-

ular species of mosquito was chosen because of its sus-

ceptibility to the virus and because of its ease of mainte-

nance in the lab-oratory.

It is honed that the results reported in this studdy wilj

not only contribute knowledyo to the basic biology of the

virus;. but also will be of use in the eventual successi;l

emplojy'enL of an integrated count rol of mosquito populations.













LITERATURE REVIEW


Although it has been known for some time that adult

mosquitoes can act as vectors for virus diseases of verte-

brates, it was not until recently that viruses were found

which were actually pathogenic to mosquitoes. Most of the

viruses which have been reported attack the larval stages

of the mosquito.

The first demonstration of a possible virus disease in

mosquitoes was reported by Dasgupta and Ray (1957) in the

larvae of Anopheles subpictus (Gras.) collected from water

pools near Calcutta, India. They observed feulgen-positive

nuclear inclusions in the anterior secretary cells of the

midgut epithelium. Initially the inclusions were small,-

but as the disease progressed the inclusions grew larger

and coalesced until the mature inclusion body obliterated

the entire nucleus. They noted that while the normal nucleus

of secretary midgut cells was 5 by 10 u, cells containing

advanced inclusions often measured 10 by 14 u in diameter.

An iridescent virus was described from larvae of Aedes

taeniorhynchus (Wiedemann) collected at Vero Beach, Florida

(Clark et al., 1965), and since that time iridescent viruses

have been described from several other species of mosquitoes.

Kellen et al. (1963) described unusual tetragonal inclu-

sion bodies in the limb buds and hypodermal cells in larvae








of Culex tarsalis Coquillett collected from Madera County,

California. The viral nature of this disease was not con-

firmed, but Clark and Chapman (1969) reported very similar

inclusions in larvae of Culex salinarius Coquillett collected

from Calcasieu Parish, Louisiana, and unpublished electron

micrographs of these inclusions strongly suggest that they

are viral in nature.

Clark et al. (1969) reported both cytoplasmic-polyhedrosis

virus (CPV) and nuclear-polyhedrosis virus (NPV) infections

in Louisiana mosquitoes. The CPV was described from larvae

of C. salinarius collected in Calcasieu Parish, Louisiana.

The disease attacked the midgut where virus inclusion bodies

ranging from 0.12 to 0.625 u could be found; the actual

virions appeared to be spherical measuring about 50 mu in

diameter. The NPV, the subject of this study, was found in

larvae of A. sollicitans collected from Cameron Parish,

Louisiana. The inclusion bodies developed in the nuclei of

cells in the midgut, gastric caecae, and, in one case, the

malphigian tubules. Measurements made from electron micro-

graphs indicated the polyhedra ranged in size from 0.1 u to

slightly over 1.0 u; the occluded rod-shaped virions measured

approximately 250 mu in length by 75 mu in diameter. This

is the only confirmed report of a NPV in mosquitoes; and the

XPV reported from larvae of the crane fly, Tipula Daludosa

(Meigen), in England (Smith and Xeros, 1954a) is the only

other report of this type of virus in a dipteran.

However, NPVs, also known by the generic name 3orr3lina-

virus, have been reported from approximately 200 species of

Lapidoptera and Eymenopterc (Aizawa, 1963). Bergold (1963)








described the genus Borrelinavirus as causing the development

of polyhedral nuclear inclusions in the larvae of Hymenoptera

and Lepidoptera. He stated that the polyhedron-shaped pro-

tein inclusions range in size from 0.5 to 15 u in diameter

and that they commonly crystallize as dodecahedra, tetra-

hedra, or cubes. The virus particles, which may be occluied

singly or in bundles in the protein matrix, are rod shaped and

range from 20 to 70 mu in diameter by 200 to 700 mu in length.

The rods are bounded by two membranes: the developmental or

outer membrane and the intimate or inner membrane.

The external symptoms of larvae afflicted with NPVs in-

clude discolorations of the integument and other tissues,

including the hemolymph; sluggishness; and loss of appetite.

Aizawa (1963)states that in the Lepidoptera,polyhedra are

formed in the nuclei of blood cells, fat body, tracheal ma-

trix, and epidermis, while in the Hymenoptera the nuclei of

midgut epithelium cells are the sites of polyhedral formation.

In the crane fly, T. paludosa, Smith and Xeros (1954) found

that the polyhedra developed in the nuclei of fat body and

blood cells.

More recent reports by many workers confirm the results

of the previous investigations. Reporting on a mixed NPV

infection in the cabbage looper, Tricoplusia ni (Hubner),

Heimpel and Adams (1966) found polyhedra containing bundles

of rods enclosed in a double membrane in the nuclei of cells

in the midgut, fat body, hypodermis, and tracheal matrix. A

second type of virus,forming smaller polyhedra and containing

only single rods,was found in nuclei of the hypodermis,








tracheal matrix, and, occasionally, in the midgut. Adams

et al. (1963) described a new 2?V from the zebra cater-

pillar, Caramca ic icta (err.), in which virus rods were

found occluded, both singly and in bundles, in nuclei of

the fat body, tracheal matrix, and epidermis. Kislov et al.

(1969) reported polyhedra in the hemocytes of the Egyptian

cot onworm, Spodc-er 4 littoralis (3oisduval). Tanada et al.

(1969) described a new strain of a N?V which caused exten-

sive cellular hypertrophy iq tracheal cells of the armyworm,

Pseudaletia uniouncta (.awor-h).

Benz (1960) described in detail the histopa-hological

changes which occurred in the midgut of the sawfly, D:rion

hercyniae (Hartig),infected with a N?V, and Smirnoff (1968)

reported a new NPV virus attacking the midgut nuclei of the

mountain ash sawfly, Pristiphora geniculata (Geoffr.)

In general, the sequence of events which takes place

once a cell has become infected is the same irrespective of

the species of insect or tissue attacked. Xeros (1956) was

one of the first workers to conduct detailed investigations

into the cytological changes which take place in infected

cells. Studying fat body and midgut of Lepidoptera and mid-

gut of Hymenoprera, infected with NPVs, he found that a pro-

teinaceous network which he termed the virogenic stroma had

formed de novo in the center of infected nuclei. .s this

virogenic stroma grew, it became increasingly feulgen-positive;

fne viral rodlets, initially 6 by 120 mu, grew within the

vesicles of the stroma to about 28 by 280 mu, after which









they were released into the ring zone, which is the area

between the virogenic stroma and the nuclear membrane. In

the ring zone the rods acquired still growing capsule mem-

branes which deposited a capsule protein around the rods,

which were then occluded in crystalline polyhedra. He

reported that in the late stages of infection the polyhedra

grew in the enlarged vesicles of the' virogenic stroma and

that this network eventually atrophied.

Evidence supporting the development of a viral rod form-

ing virogenic stroma in nuclei infected with NPVs has been

reported by Benz (1960) in studies on the sawfly, D. hercy-

naie; by Benz (1963) in stidies on the moth, Malacosoma

alpicola (Staudinger); by Morris (1966) in studies on the

western oak looper, Lambdina fisillaria somniara (Hulst.);

and by Morris in autoradiographic studies on D. hercyniae

(1968). These workers found that DNA, RNA, and protein syn-

thesis increased in the early stages of infection, and a

nuclear virogenic stroma was formed which became increasingly

feulgen-positive. As polyhedra formation began, nuclear and

cytoplasmic RNA levels decreased, but they were always higher

than R.A levels in uninfected cells. Benz (1963) observed

that the nucleoli multiplied, and often persisted throughout

polyhedra formation, most likely to continue production of

R NA for the synthesis of polyhedral protein.

Shigematsu and Nogouchi (1969a, b, c) further supported

those observations. They conducted detailed time-course

radioactive tracer studies on the synthesis of nucleic acids








and proteins during the multiplication of a NPV in the

silkworm, 3ombyx mori Linnaeus. ,hey fou-d peaks of DNA

synthesis at 4 hours after infection, wnich they believed

tc be replication, viral DNA, and at 25 hours after infection,

which they did not explain. In addition, they observed two

main peaks of protein synthesis: one at 25 hours afwer treat-

ment, which they thought to be viral protein, and the other,

25 hours after the first, which they believed to be polyhedral

protein.

In electron microscope investigations of NPV infections

in the midgut cells of D. hercyniae and Neodicrion oratti

(Geoffr.), Bird (1957) described a process somewhat different

from that proposed by Xeros (1956). Bird found that upon

gaining entry to a nucleus, rcd-shaped virus particles

attached to host chromatin and converted it into minute

spherical bodies surrounded by membranes, which increase in

size to form new viral rods. He suggested that the newly

formed virus could escape from its outer membrane and repeat

Sthe cycle. Rods or spheres which were occluded in polyhedra

ceased to develop any further. Bird (1964) described pro-

cesses similar to this in a study of a NV infection in the

spruce budworm, Choristoneura funiferana EKbn.).

Day et al. (1958; studied the structure and development

of a NPV a-fecting the larvae of the moth, Pterclocera

E.mplicornis Walker. They reported that the rods were abun-

dant in masses of chror.ntin and that most likely they mrlt-

plied there. Although hey studied zhe process of vi;ra








replication in detail, they stated they could not be certain

of the actual events of viral multiplication. After the rods

were formed, they were released into the nucleoplasm where

they were enclosed in membranes, and, eventually, occluded

in polyhedra. They also found circular objects in the nucleo-

plasm which they suggested were developmental membranes from

which viral rods had been released to initiate further cycles

of replication. They noted that fully developed viral rods

apparently acted as sites for polyhedral protein crystalli-

zation, and that these rods were occluded randomly. They

found no evidence for further development or maturation of

the rods once they had been occluded.

Xrieg and Huger (1969), studying the formation of NPVs

in Galleria mellonella (Linnaeus), Lymatria dispar (Hbn.),

and Choristoneura murinana (Hbn.), found that there were

different modes of virogenesis within the same disease. In

some cases, naked rods or long filaments were surrounded by

membranes which they believed to be developmental membranes.

In another type of development, they found spheres which

were limited by double membranes and were derived from tne

virogenic stroma. They believed these spheres contained

virogenic material and were capable of giving rise to fully

developed viruses.

Smith (1955) described the unusual behavior of the NPV

of T. Daludosa, which attacks the nuclei of fat body and

blood cells. He found that a thin-walled vesicle forced

around rods whic- were produced in dense chromatic masses





12


in the virogenic stroma and that a fluid (possibly a protein)

suspended the roc in this membrane. After leaving -he strcma,

a large number of these vehicles collected on the nuclear

membrane, where another membrane apparently enveloped masses

of them. The mass of rods condensed and eventually the

vesicles flattened against the viral rods. At this stage,

the entire capsule appeared to be much smaller than in i-s

initial state. Smith stated that -he process continued

and by this process crescent-shaped polyhedra were forr-ed,

and that the arrangement of viral rods in the polyhedra was

random.

Xaros (1966) studied the same disease in the blood cells

and found that infected cells underwent a period of extensive

proliferation after which a normal virogentsis occurred.

During division, the telophase chromosomes became attached

to the nuclear membrane, a virogenic stroma arose in the

ccener of the nucleus, and viral rods were produced in the

cords of this stroma. He found that the capsule membranes

were acquired only after the rods lef- the stroma.

T.e physical and chemical properties of polyhedra have

been extensively studied by Sergold (1S47) and MIorgan et al.

(1955, 1956) and recently reviewed by Bargold (1963). Ber-

gold stated that polyhedra are composed of large spherical

protein nolacules which vary from 200,000 to 400,000 in

molecular weight, depending on the species of virus. Thecs

large molecules ar apparently arranged in a face-concered

cubic lattice, which imrpl;c the molecules are not in ;he








closest possible arrangement, suggesting there are possible

points of attraction. Polyhedra dissolve in NaOH, KOX, '-H

H2SO and CE3COCH, but not in organic solvents.

Xeros (1966) found that the polyhedra of the NPV attack-

ing T. paludosa elongated when subjected to Carnoy's fixative

or to acetocarmine, but not when placed in cosmic acid. These

polyhedra had several unusual properties; they exhibited a

to 16 u bands at 1/2 u intervals; there were cortices which

stained less intensely with feulgen than other areas in the

center of the polyhedra, suggesting these areas may be free

of virus rods; and the crystalline lattice oG the polyhedra

was not resolved. Xeros also found that young polyhedra

were much more sensitive to acids and alkalis than were old

ones, especially those taken from dead larvae.

The fine structure of NPVs was first investigated by Ber-

gold (1950) and since then has been studied by many workers.

Initially, Bergold postulated that an outer membrane was

formed, upon which viral spheres developed, and he named

this the developmental membrane. Inside this membrane there

existed a second membrane which was termed the intimate

membrane (Bergold, 1952). Bergold (i963) later presented

schematic models for the NPVs of 3. morl in which rods were

enclosed singly in a developmental membrane, and aphyca-.

frugiperda (Smt.), in which four rods were enclosed within

a common developmental membrane. The 3. mori model showed

a dense ccntr-l core mado uo of 8 subunit; approximately .C

mu in diameter. he time i te membrane was- spaced away from








the core at 6 rmu and was 4 .u thick. Following this men-

brane was another spacing of 6 mu,after which the 7.5 mu

thick developmental mr_.brane was found.

On the basis of disc-shaped zoherical subunits obtained

by alkaline degradation of whcle virus particles, and helical

structures seen on uncgradd virus particles, l;rieg (1961)

proposed a model for :P:Vs similar to tobacco mor.cic virus.

n the Krieg model the disc-shaped subunits, in which he

believed the DNA was located, were 50 mu in diameter and

5 to 10 mu thick, with a 10 to 20 mu hole in the center.

Harrap and Juniper (1966) found a banded structure en

the intimate membrane of a N-V attacking the larvae cf the

tortiseshell butterfly, Aglais urtica4 (7ab.), in negatively

stained preparations. This banded structure was 45 A in

width, which compared favorably with the Krieg model. Koz-

lov and Alexeenko (1967) proposed a model similar to both

Bergold's and Krieg's, only they believed the DNA to exist

in a twisted central core rather than subunits. However,

this model cannot be taken too seriously, as it is mos:

likely based on artifacts as shown by Gregory ez al. (1969).

:i.meno ea al. (19S6) also studied the KPV of 3. nori. They

found that the outer mernbrane had a double .membrane structure

and confirmed hc striazed structure seen on the intimate

merbrans by others. Teakle (1969) also found parallel cross-

striaticos c- the. ntimtae mermrane of the NPV attacking the

butterfly Antha:.. varia (.-,n..









While studying the virogenesis of the NPVs of G. mel-

lonella, L. dispar, and C. nurinara, Krieg and Huger (1969)

noticed massive fibrillar networks in the nucleus and cyto-

plasm. This fibrillar material appeared to be derived from

a unit membrane structure and was deposited on the surfaces

of growing polyhedra, and they proposed that this material

was polyhedral protein. Similar structures have been re-

ported by Summers and Arnott (1969) from NPV-infected tra-

cheole cells of T. ni, and by Granados and Roberts (1970)

from the fat body cells of Estigmene acrea (Dry.) infected

with an insect poxvirus.

Huger and Krieg (1969) reported the presence of spindle-

shaped protein inclusions which occur in the cytoplasm of

fat body, tracheole, and epidermal cells of C. murinara

infected with a NPV. These bodies were associated with

typical masses of polyhedral and ranged in size from 1 to

6 u in length by 1 to 4 u in width. After treatment with

0.1 N NaOH or 0.1 N HC1, they elongated to form long fusiform

bodies which eventually dissolved. It is interesting to

note that this spindle shape is characteristic of early

inclusion development in the insect poxviruses, described

by Weiser (1965), Weiser and Vago (1966), and Granados and

Roberts (1970). All of these spindle-shaped inclusions

develop in the cytoplasm.














MATERIALS AND !METHODS


Larval Roaring and Colony maintenance

A laboratory colony of Aedes triseriatus mosquitoes was

originated from adults obtained from'a strain originally

colonized from field larvae collected in Lake Charles,

Louisiana. Adults were held in 16-mesh wire screen cages,

20 x 20 x 30 inches long, and the cages were maintained in

a controlled room with a temperature of 30C, relative humid-

ity of 30 per cent, and a light to dark ratio period of 16:8.

The cages were provided with 10 per cent sucrose solutions as

a food source for males, and adult females were blooded daily

on the shaven bellies of guinea pigs. Paper cups lined with

mois : tocewling were placed in the canes daily as ovipositicn

sites.

-arqcs were dried slowly over a period of four days to

allow proper e.nbryonation. Hatching was induced by flooding

the ,Cgs ,Jith distilled water containing a small amount of

higjh potein hog supplement (0.5 gm/100 ml). T:,enty-four

hc-rs after h t':h ing, groups of 200 larvae were transferred to

i-iu .vi' dual 9 x 12 inch en;umel rparing pans and reared on a

hog "uppl'ementi'/hay infusion mediu-m (0.5 gm hog supplement,

100 i;1 h- inCuLion, 800 ni of disLilled water).

The h1y infsi an was prepared by homogi.nizing appioxiir'ately

50 i of a.lai.fa 'l:ay in 500 i.i of dLst-illed water, rnd strain-








ing the homogenate through a 100-m-sh screen to filter cut

large pieces of hay. The pans were placed in a rearing

room maintained at 300C, and aerated to prevent the growth

of bacterial scum on the surface of the water. Once pp'ation

began, pupae were removed every other day, placed in dis-

tilled water, and held in adult rearing cages for e.ergence.

Preparation and Quantificatici of Virus Inocu'.a

Virus inocula were prepared by triturating 100 patently

infected second-, third-, and fourth-instar larvae in 5C ml

of distilled water. inocula were quantified by making poly-

hedra counts with a Petrcff--ausser bacterial counter.

Inocula were diluted so that all suspensions contained the

same quantity of polyhedra, and they .are then stored in a

refrigerator at 4002.

Znoculattor of Larvae

Normally, suspensions prepared and stored as outlined

would have very low infectivity, since the majority of the

viable virus would be occluded in the polyhedra. In order

to achieve greater infectivit,, viral rods were liberated

by dissociating the polyhedral protein prior to inoculation.

This was accomplished by placing 1 ml of the polyhedra sus-

pension in 40 ml of 0.035 M Na CO3/0.05 M NaCl adju-ed to

pH 10.9. The solution was allowed to sand at room temper-

ature for two hours, after which the p. was readjusted to

7-7 1/2 with 0.01 N KEC. Inocula for all larval treatment.

were prepared in this mannr.







Larvae were inoculated in groups of 200 by placing. theom

overnight in j0 ml of inoculum as prepared above. -he fclelw-

ing morning the larvae and inoculum were transferred to pre-

pared rearing trays.

mortalityy Studies

To obtain dos-ge-mortali y data, groups of larvae were

inoculated at 24, 48, 72, and 96 hours after hatching. The

larvae were reared as described above until they reached the

desired age, at which time they were collected, exposed to

virus, and then placed in freshly prepared media. When pup .

tion began, the pupae were collected and counted every other

day, and mortality rates were computed on -he basis of total

pupation. Control larvae were treated identically e cept

that the 1 ml of polyhedra suspension was deleted from the

procedure.

Trancovar=.a rransmi:sion Stucis3

Three replicates were macd wiTh larvae inoculated at 24,

42, 72, and 96 hours of age. Mortality rates were ccr.putr

as described in -he previo-s tes-. the pupae were then

pooled and allowed :o emerge in clean adult holding cages.

Adults were fed, blooded, a.d egead,as described above, and

controls were handled in the sa-i manner but maintained in

a separate caga. Eggs were collected over a period of two

:weeks, hached, and the larvae were reared normally, 200

larvae per tray. Rearing trays were examined daily for

p-ecntly infected larvae, -nd final morality rates ware

cCmpured on the basis of to-al upation.








Another test., similar to the previous one, was conducted

in which three replicates also were made with larvce inocu-

lated at 24, 43, 72, and 95 hours after hatching. In this

test pupae were not pooled but maintained in separa-c ccn-

tainrs. Puoae which did not emerge were squaeh.-d on c gl-_

slide in a drop of distilled water and examined by p..ase

microscopy for the presence of polyhedra. CThose pupa \.:.c..

were found to contain polyh'dra were subtracced fron. cz a ;t _

pup-tion rate for their respective test grcrp. Sex ra__

were recorded for the adults which emerged,and samples of

adult females from each repiLcate were squashed and examrind

for virus inclusion-.

Histoloqy E-d C7Zopatholoc,

Licht Mis oscony

The larval mortality te-.s served a- sources of infected

Larvae for ..11 o-cer investigations. Larval rearing pans

were exu.lined daily..and par of the larv a which showed patent

ineectorns was collected and fixed in e-th _- C.rnoy's fixative

for two to three hours, or in aqueous Boain's for several

ia"s. The larvae were then deh1ydrated by passing them

through an ascending series of ethanol, sue massively infil-

tra-ced with tertiary butanol and t-butanol/Paraplast mix-

tures, and e.bedfded -n pure Paraplast. Sections approxi:.atel

6 u t.-ick were cut on a Lei-oz microtome and stained by one

of the following procedures: Eelafield's hamaooxylin and

ecoin, Heidanhain's hematoxylin, Har..'s stain for poly..-era,



Scientific Products, bvanston, Illincis








or with t.a feul-aon reac--ion for D,.. (Appendix i).



infectedd and healthy rv.:o ;era c_.t into .nall 1 .

pieces a fixed for Zhro hours ih 3 2' r cent c. uto:=-

hyde in 0.1 -2: p-.os_-te buff: .. :hen transferred to C.1

- phosphate buffer ovcrnighc. '- following a t.e pieces

of tissue were post-fixed with 1 oer cent 0sO, in.1

.phosohat buffer, de-hdr-t.... by passage thrcac incrsi

concentrations of e-hancl to propyle:.a oxide, and caecdiad

i an c on-araldi- e mixture (::olale-ha.ar, 15-6). in so

cases the midgut and gastric caacae v.re dissected from

individual larvae in 0.1 phosphate buffer and carried

through the same procedure. Ultra-thin sections were cut

on a Sorvall MT-2 microtome wi-h glass knives and then

sai.ed with saturated uranyl acetate, followed by load

citrate (Venable and Coggeshall, 1965). The sections were

examined and phorographed with a Hitachi 15-E electron

microscope, using accelerating volcages of 50 and 75 KV.

Scanninc Electron Mic'oscov

The midgut and gastric caacae of patently infected lar-

vae were dissected in distilled water, transferred -o a

drop of distilled water on a glass covered metal stub, and

triturated with forceps until no large pieces of tissue

remained. T.e water was allowed to evaocrate and the speci-

..cs were than transferred to tne small rotating table in

a D.o.-on JV-502 hiCh vacuur:m evaorator where t w ooa.-c-

wrih 20C-300 A of gold a.t a v.,uum of 2 x 10 -or:. S0ci-..:aa







21


were examined .ith a ambridge Steroscan electron .-.icrcsco. e

at accelerating- voltages -- 5e an lC :V.

Freeze-2tc'ing

The midget and g;strc caece? cf pate-.mly infected lar-ae

were remrroved in 0.1 M phosphate buffer, fixed with 3 prc

cent gluteraldehyde in 0.1 :1 phcsphate buffer for 13 minutes

and tra-sferred to a 30 per cen-a glycerol solution over-ig...

to prevent the formation of ice crystals. The following >.,

the specimeans ware centered 3...c 3 mr, brass plachsts, rapidly

frozen in :reon-22 (-1530C;, an. stored i. lquid nisrccge

(-1-~uC) until ready for use. Individual. specin0 .s wer-

then place on a precooled specimsn sta-e (-IC0'C) in ,

Balzer's freeze-stch microtome and fractured at this tL:-- er-

ature under a vacuum less tan 2 x 10-6 orr. The fractured

surface was then etched for two minutes, -'plicated, and

shadowed with placinum and carbon. Tne replicas were

cleaned by washing -the. for one hour in ChloroxR, followed

by an hour in distilled wa-er, after which they were examined

and photographed with a Hitachi 125-E electron microscope at

accelerating voltages of 75 and 100 XV.

Measure.ment of Viral Comoonents

All measure.ents of viral coc=onents were made directly

from electron r.icroscope negatives.

Cher.icl behavior of Polvhedra

T. mridguts and gastri; caacae of heavilyy infrctcd -cird-

and fcurth-insear larvae were removed and placed on a slide

in a drop of distilled water, covee- with a co.'r slip, ...





22


gently squashed with thumb pressure. A drop of one of the

following solutions was placed on the edge of the cover slip,

and subsequent changes in polyhedra were observed with phase

microscopy. The solutions used were 1 N, 01. N, 0.01 N and

0.001 N NaOH, glacial CH COOH, and 1 N, 0.1 N, 0.01 N and

0.001 N HC1.














RESULTS


Pathology

Gross Pathology

The initial signs of infection began to appear 48 to 72

hours after larvae had been placed in the inoculum. In

trials in which all larvae were inoculated at the same age,

the most conspicuous sign of early infection was size differ-

ence. In such trials, most of the non-infected and control

larvae were advanced at least one instar beyond infected

larvae within 48 hours after inoculation. However, diag-

nosis on the basis of size alone could not be used as the

definitive criterion of infection as there were always

healthy larvae with slower than normal growth rates. This

was particularly true in trials where larvae were inoculated

at 72 hours of age or older.

Infections were confirmed by examining larvae suspected

of being diseased under a dissecting scope against a black

background. Within 48 to 72 hours after inoculation, the

nuclei in the stomach and/or the gastric caecae of infected

larvae appeared as hypertrophied white spherules. These

enlarged infected nuclei could be easily observed by viewing

the midgut through the intersegmental membranes. The opaque

white color was attributable to advanced stages of polyhedra

formation within these nuclei. Although the full length of

23







the midgut epithelium often showed these hypertrophied nuclei,

most larvae in this stage of infection behaved as healthy

larvae, feeding normally and moving throughout the media

without any apparent difficulty. The midgut epithelium of

control larvae was devoid of such symptoms. In most cases

this tissue was translucent when viewed against a black back-

ground, although occasionally the midgut appeared milky

white. This may have been the result of a nutritional

irregularity.

As the infection progressed, the larvae became sluggish

and suffered a marked loss of appetite. Their movements

along the surface of the water were much slower than those

of healthy larvae, and frequently they lingered in one area

for several minutes, behavior very atypical of healthy larvae.

Eventually the entire midgut epithelium became an opaque

white, and in many cases this tissue hypertrophied to a

point where it displaced most of the hemocoel. Larvae with

such heavy infections rarely moved unless disturbedand

death usually followed within a few hours after such a con-

dition developed. Dead larvae eventually sank to the

bottom of the rearing pans whether mortality occurred on

the surface or not, and carcasses which were not removed

from the pans were cannibalized by other larvae.

Stomachs and gastric caecae of both living and patently

infected and recently deceased larvae, when dissected in

distilled water, squashed, and examined with phase micro-

scopy, revealed the presence of numerous highly refractile








inclusion bodies. These inclusions varied widely in size and

shape, including small spheres 0.5 u in diameter, slender

0.5 x 5 u stick-like and club-like formations, and large

rough-edged spindle-and ellipsoidal-shaped bodies, 3 to 6 u

in diameter by 6 to 20 u in length.

Histopathology

Histological examinations of sectioned material indicated

that all cells of the cardia, gastric caecae, and stomach

were susceptible to infection (Figs. 1, 2, 3, and 4). Poly-

hedra were never observed in the nuclei of any other tissues

no matter how heavy the infection in the midgut epithelium.

However, this does not exclude the possibility that viral

replication may have occurred in other tissues.

Apparently some of the first cells to become infected

were those at the base of the cardiac (Fig. 5). The infection

then spread to the gastric caecae and further down the mid-

gut, although not necessarily in that order. In many cases

heavy infections could be demonstrated in the nuclei of

stomach cells just anterior to the junction of the midgut

before any of the nuclei in the gastric caecae showed signs

of infection. The infection apparently was spread by the

sloughing off and breakdown of infected cells, although this

was not the fate of all infected cells. The vast majority

of the infective material from disrupted cells, consisting

of free virus and polyhedra, traveled between the microvilli

of the epithelial cells and the peritrophic membrane. A

gradual movement of the infective material throughout the gut

was produced by the peristaltic actions of the gut.




















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In general, infected nuclei began to hypertrophy about

24 hours after infection. The host chromatin first appeared

to move to the nuclear envelope, after which a typical viro-

genic stroma developed in the center of the nucleus (Fig. 6).

In sections stained by the feulgen reaction for DNA, the

stroma was weakly positive. In nuclei in which polyhedra

had begun to develop, the reaction was also positive, but

weaker than in cells with less advanced infections. Poly-

hedra initially developed in the ring zone and later,as

the virogenic stroma degenerated, throughout the entire

nucleus (Fig. 7). In fresh squashes of infected midgut,

small polyhedra were observed developing throughout the

stromatic area, although the largest concentrations were

always found towards the periphery of the nucleus. Nuclei

which contained mature polyhedra often showed heavy concen-

trations of chromatin on the nuclear envelope. The nucleolus

multiplied during the period of viral replication and in

many cases nucleioli persisted throughout polyhedral form-

ation.

Infected cells which had been sloughed off were replaced

by regenerative cells situated on the basement membrane.

These cells also became infected, usually while they were in

the process of developing to mature midgut cells. In some

instances groups of cells became infected at approximately

the same time. As they were sloughed off, the regenerative

cells replacing them became infected. Sections through such

areas gave the impression of a proliferation of cells










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characteristic of tumor-like growths (Fig. 8). Just how

frequently this occurred was not determined, but it seemed

relatively rare.

In very late infections, when the hypertrophied stomach

and gastric caecae occupied most of the body cavity, the

tissues of the fat body and muscles were also atrophied. At

this stage, sections through polyhedra in the nuclei of

infected tissues revealed them to be spheres, spindle, or

ellipsoids, depending on both the actual stage of maturity

and the angle at which they were sectioned. Hamm's stain

gave the best results for realizing infected cells and poly-

hedral shapes, although Delafield's hematoxylin and eosin

often produced very good results.

Virus Entry into Cells

In the midgut of control larvae, the peritrophic membrane

was situated close to the microvilli (Fig. 1), and the space

between the microvilli and the peritrophic membrane was

filled with digested material. In infected larvae, this

space gradually increased, and the majority of the additional

material filling this space was of viral origin. The viral

nature of this material was demonstrated by both light and

electron microscope examinations (Fig. 9-16).

The infected epithelial cells, sloughed off in the manner

described earlier, were the major source of viral material

for the spread of the infection. Once in the lumen between

the microvilli and the peritrophic membrane, these dead cells

and their organelles, including the nuclei, were rapidly















Figure 9. An oblique section through the midgut of an
infected third-instar larva. The red-staining
material between the peritrophic membrane and
the epithelial cells is largely viral in nature.
Stained with Heidenhain's hematoxylin, 250 X.


Figure 10.


An oblique section through the infected midgut
of a third-instar larva. The red-staining
material both in the cells and in the space be-
tween the peritrophic membrane and the epithe-
lial cells is polyhedral protein. Stained with
Hamm's stain, 350 X.














I. .





ItjI





)l1)

'I.'
* '4~. i
4 '- -
nr











A polyhedra dissociating in the midgut lumen.
Freeze-etch replica, 18,000 X.


A polyhedra dissociating between the peritrophic
membrane and the microvilli. Ultra-thin section,
26,000 X.


Virions accumulated along the peritrophic mem-
brane at the site of its formation. 16,000 X.


Polyhedra and free virus in the area between the
microvilli and the peritrophic membrane in the
stomach. Note how much more condensed the peri-
trophic membrane is in this area of the midgut.
12,000 X.










Virions in an area between the peritrophic mem-
brane and the microvilli of the stomach. Note
the retracted intimate membranes. 47,000 X.


Two virions "attached" to microvilli in the
stomach. 60,000 X.


Figure 11.



Figure 12.


Figure 13.



Figure 14.


Figure 15.




Figure 16.










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digested by the hydrolytic enzymes of the gut. Polyhedra,

in various stages of formation, began to dissociate once

released from disintegrating nuclei, liberating virus rods.

Also, numerous non-occluded rods were released immediately

with the disintegration of a nucleus. Viral rods, devoid of

developmental membranes but which still possessed what ap-

peared to be a retracted intimate membrane, were the most

common form of virus observed in this released material

(Figs. 13-16). Virions with retracted intimate membranes

were frequently seen near the brush border and in some cases

appeared to have attached to the microvilli (Fig. 16). How-

ever, no stage or component of the virus was ever observed

actually entering the cytoplasm or the nucleus of a cell.

Apparently, the virtually naked viral DNA is injected into

the cytoplasm and in some manner eventually enters the

nucleus where it replicates.


Cytopathology

Replication and development of the Virus

A healthy gastric caecum or stomach cell (Fig. 17) was

characterized by a large nucleus, averaging 10 u in diameter,

with polytene chromosomes. Electron micrographs of such

cells revealed the chromatin clumped together (most likely

cross-sections through chromosomes) in certain areas through-

out the nucleoplasm. The nucleolus was most commonly found

near the center of the nucleus and was somewhat denser than

the surrounding chromatin. The cytoplasm of healthy cells








had an abundance of small tube-like mitochondria, several

medium-sized mitochondria, and an occasional large mito-

chondrion. The number of ribosomes and the amount of rough

endoplasmic reticulum varied depending on whether the cell

was located in the gastric caecae or in the anterior or

posterior stomach.

Infected cells varied in size, but in general they were

characterized by hypertrophied nuclei averaging 12 to 20 u

in diameter. Occasionally infected nuclei were seen with

diameters in excess of 30 u.

In the early stages of infection, all host chromatin

except the nucleolus was broken down, forming a diffuse,

amorphous mass of nucleoprotein. The nucleolus underwent

several divisions, although the final number of nucleoli in

any one nucleus was never determined (Fig. 18). At this

stage, the chromatin was discernible because of its slightly

denser staining properties than those of the surrounding

material. The chromatin gradually reggregated in the center

of the nucleus and formed a virogenic stroma (Fig. 19). As

the "cords" of this stroma became more distinct, nucleocapids

were always associated with them, and were usually either

closely adjoined to the chromatin or found in the spaces be-

tween the chromatic cords (Fig. 20). Careful examination of

these same areas occasionally revealed the presence of dense

staining helical coils which may have been newly formed

viral nucleoprotein (Fig. 21). However, these coils were

rarely observed and were probably artifacts.










Figure 17.



Figure 18.












Figure 19.


The "cords" of a well-developed
14,000 X.


virogenic stroma.


A helical coil typical of those occasionally
associated with the virogenic stroma. 90,000 X.


Capsids in an early stage of formation. Note the
single capsid to the right of the aggregation of
capsid protein. 46',000 X.


A healthy midgut epithelium cell from the stomach
of a third-instar larva. 3,200 X.


An infected nucleus in which the nucleolus has
moved to the edge of the nuclear envelope and
begun dividing. The virogenic stroma has not yet
developed. 5,000 X.









A nucleus in an early stage of infection with the
virogenic stroma easily visible in the central
area. The small dense rods are newly formed
nucleocapsids. 5,000 X.


Figure 20.


Figure 21.



Figure 22.










P~ I-..'


I '. p l,





IAt.


03~t


S A'


21


".


e)


,.




Yil
I
I~
)

"







The spaces between the cords were also the observed

sites of capsid formation (Fig. 22). Tubular capsids ap-

peared singly or in parallel aggregations containing any-

where from 2 to over 100 capsids per aggregation (Figs.23

and 24).

The dimensions of developing and complete viruses, and some

of their components, are presented in Tables 1 and 2. Capsids

in early stages of formation had a "coat" thickness of about

9 mu (Fig. 22), while empty capsids,either singly or in aggre-

gations, averaged 35 mu in diameter, with the lumen measuring

20 mu. Measurements made of the capsid "coat" from further

developed capsids indicated the constituent protein had further

condensed to about 7.5 mu in thickness. The length of the

capsids varied from aggregation to aggregation but was rela-

tively constant within any one.

Nucleocapsids apparently formed when the viral nucleo-

protein entered the tubular capsids, either singly or while

they were in aggregations, but the actual process of entry

was not observed (Figs. 25, 26, and 27). Aggregations of

nucleocapsids were also observed in freeze-etch replicas

(Fig. 28). It is interesting to note that the diameter of

the empty capsids was less than that of the complete nucleo-

capsids (Table 1). The completed nucleocapsid consisted of

a dense nucleic acid core, 38 mu in diameter, enclosed in

a capsid 5 mu thick. The overall nucleocapsid diameter was

approximately 48 mu, while the length averaged 184 mu, but

varied widely.










Figure 23.



Figure 24.











Figure 25.



Figure 26.











Figure 27.



Figure 28.


Cross (top) and longitudinal bottomn) sections
through two ordered arrays of copsids. 46,000 X.


A cross--section through an aggregation of cap-
sids. Note that a few of the capsids contain
nucleic acid. 70,000 X.









A cross-section through an aggregation of newly
formed nucleocapsids. Note the capsids sur-
rounding the dense central core. 340,000 X.


An aggregation of newly formed nucleocapsids:.
Note the empty capsids within the aggregation.
140,000 X.










A cross-section through a single nucleocapsid.
The capsid is easily seen around the dense cen-
tral core. 250,000 X.


A freeze-etch replica of an infected nucleus.
The arruo:s indicate aggregations of nuclco-
capsids. 10,500 X.





L












































'* U




A': .1


'24 ,.


: ,

l1* "'?
' "
,


SI .28


I~ ~7


-~. '~i~


-cllf


-r. I
IlL


I
SA
.















Table 1.--Dimensions of various viral components during virus
assembly (average dimension in mu)


Capsid Capsid Capsid Nucleocapsid Nucleocapsid Capsid
Diameter Lumen Coat Diameter Core (Visible
Thickness Diameter Outisde
Core)

35.3 20.5 7.5 48.2 38.2 5.0











Table 2.-Dimensions of non-occluded and occluded whole virus
(in mu)


Whole Virus Nucleocapsid Whole Virus Nucleocapsid
Diameter Diameter Length Length

Non-occluded 79.4 39.4 236.0 184.0
virus

Occluded 63.6 38.5 200.8 157.1
virus








Sections cut along the longitudinal axis of some of the

nucleocapsid aggregations indicated that at least in some

cases the nucleoprotein entered elongated developing capsids

from which complete nucleocapsids of a fairly uniform length

would eventually "bud" off (Figs. 29 and 30). In many nuclei

this process was never observed, while in others it was com-

mon. Figure 31 is typical of a nucleus in which the majority

of the nucleocapsids are being formed in elongated aggregations

rather than singly. In this particular section an unusually

large number of nocleocapsids were found in each aggregation

and the deposit of chromatin on the internal surface of the

nuclear envelope was unusually heavy. The nucleus of the

adjacent cell was in a similar condition.

Frequently, long, and, in some cases, curved nucleocap-

sids were observed, measuring up to 1 u in length (Fig. 32).

In other nuclei, especially those with early infections, short

nucleocapsids 50 to 70 mu in length were not uncommon. Al-

though the length of these unusual nucleocapsids varied con-

siderably, the average diameter of 50 mu was fairly constant.

Intimate and Outer Membrane Formationl

The development of these two membranes i8 considered to-

gether because there was an intimate, though not clear, re-

lationship between their formation.

After a nucleocapsid had been formed in one of the manners

described above, it acquired an intimate and an outer



1. In standard virus nomenclature the intimate membrane
would be referred to as the second concentric layer of the
capsid; the outer membrane as an envelope.












Figure 29.




Figure 30.
















Figure 31.


A section along the longitudinal axis of a nucleo-
capsid aggregation. Note the uniformity in size
of the smaller nucleocapsids. 20,000 X.


A section along the longitudinal axis of a nucleo-
capsid aggregation in which the shorter nucleocap-
sids give the impression of having "budded" off
the larger nucleocapsids in a longitudinal direc-
tion. 65,000 X.












An infected nucleus cut at a plane in which the
majority of nucleocapsids are in parallel aggre-
gations. 10,000 X.


Figure 32. An atypical long and curved nucleocapsid. 52,000 X.








Figure 33. Vesicular material from which the outer and inti-
nate membranes are derived. 30,000.


Figure 34. A single nucleocapsid attached at one end to a
small vesicle. 120,000 X.






I!












P.I
Li


J





.. 4


r-



1.


'al


f AL


33


..




'* .*..


i34


r '.
w.. c.


o*i
.*..


4~


~"


. -r









membrane, apparently at about the same time. The process,

which took place throughout infected nuclei, began when a

newly formed nucleocapsid attached, either on end or along

its longitudinal axis, to a preformed amorphous vesicular

structure, the "walls" of which averaged 20 mu in thickness

(Fig. 33). Once the nucleocapsid had attached,it was gradu-

ally enveloped by this structure (Figs. 34-40). As the pro-

cess of envelopment neared completion, two distinct layers

of organization could be discerned in the segments of the

vesicle most closely applied to the nucleocapsid. The inner

layer, the one directly adjacent to the nucleocapsid, was

homogeneous in appearance. As envelopment continued,it became

evident that this was the intimate membrane. The outer layer

contained distinct areas with a unit membrane-like structure,

but it was not until the process of envelopment was complete

that this structure, the outer membrane, was readily apparent

(Fig. 38). In fully developed virus rods the unit membrane-

like structure of this outer membrane was even more apparent

(Fig. 41).

The size of these vesicles varied widely as did the number

of nucleocapsids which underwent envelopment at any one time.

Cross-sections through these vesicles revealed from 1 to 12

nucleocapsids in one plane, implying that a significantly

greater quantity of them may have been involved in envelop-

ment, especially in the latter case.

From the range in the number of nucleocapsids found with-

in any one vesicle it seems likely that the process may begin









Figure 35.




Figure 36.











Figure 37.



Figure 38.












Figure-39.



Figure 40.


Attachment and envelopment nucleocapsids. Note
that the virions within the vesicle are completely
enveloped. 100,000 X.


Envelopment of nucleocapsids and formation of the
intimate membrane in-small vesicles. Note the
vesicular material in the arc of these two vesicles.
60,000.








Envelopment of nucleocapsids and formation of the
intimate membrane in large vesicles. 61,000 X.


Longitudinal nucleocapsid envelopment in which the
unit membrane-like structure to the outer membrane
is apparent (arrow). 250,000 X.


An area where the majority of the nucleocapsids
have been enveloped individually. 40,000 X.




i




I


large


Vesicular envelopment' of-nfcleocapsids in
vesicles. 62,000 X.'







,J- IIF~


#1 T


6I


e~31~


IvY




,5 :i;~
--


5~Th


38


IK


It
r .3


?s9


~
.ti *
":~i~


,~r -


ji+ :LrS~ .i
Cr,
t,







with the attachment of one nucleocapsid to a small vesicle

and continue with an evolution of this structure by the

addition of more nucleocapsids and membrane-forming material

to a larger vesicle containing many nucleocapsids in differ-

.ent stages of envelopment.

The process by which the enveloped viruses were released

from these vesicles was obscure, but it appears that at some

point the vesicles disassociated, thereby freeing the

"completed" viruses.

In some cases it appeared that intimate and outer mem-

branes may have formed in the immediate absence of nucleo-

capsids. Empty spherical structures with membrane-like com-

ponents were occasionally seen free in the nucleoplasm in

ultra-thin sections (Fig. 41), and similar structures were

seen in freeze-etch replicas (Fig. 42).

Virus Occlusion and Polyhedra Formation

Examinations of ultra-thin sections and freeze-etch

replicas of infected nuclei indicated that the outer membrane

of complete virus rods was the crystallization site for

inclusion body protein. Once initiated, the process of crystal-

lization proceeded, causing the formation of small inclus-

sions containing from onto only a few viruses. Inclusions

formed in such a manner would either coalesce, forming larger

inclusions, or they would continue to grow individually,

gradually occluding more viruses. Only complete viruses

which had both intimate and outer membranes were occluded,

and the arrangement of these rods within the polyhedra was

random. (Figs. 43, 44, and 45).








Figure 41.






Figure 42.










Figure 43.












Figure 44.


Figure 45.


A section through an infected nucleus illustrat-
ing complete virus rods before occlusion. The
arrows indicate rods where the unit membrane-
like outer membrane is most easily resolved.
Note also the circular membraneous objects which
do not contain nucleocapsids. 47,000 X.


A freeze-etch replica of an infected nucleus.
The circular structures on the left-hand side
of the figure appear to be empty membraneous
structures similar to those seen in thin sec-
tions. 48,000 X.







Freeze-etch replica-of a developing polyhedra.
Note the bulbous end on some of the rods, indi-
cative of membranes in the process of condensing.
30,000 X.









A freeze-etch replica of a developing inclusion.
The arrow indicates the suggested direction of
the lattice. 30,000 X.


An ultra-thin section through a developing poly-
hedra. The membranes in the section are more
condensed than those in free viruses. Careful
examination of the material in the upper left-
hand corner of this figure will reveal the pres-
ence of membraneous disc-like structures which
vary in size. 40,00, X.



u








-31


-??*
r4tf ^
( ."
F K'^


V 9
,bI~ eg


0.~


F~2~


45P.
r I


-."Z'


'AlM\.;








Measurements (Table 2) of viral rods made before, during,

and after polyhedra formation indicated that the rods under-

went a gradual reduction in size, apparently involving a

further condensation or tightening of the intimate and outer

membranes around the nucleocapsid. The process of conden-

sation usually began at one end of the rod and gradually

proceeded to the other end. For this reason many occluded

rods examined, both in ultra-thin sections and in freeze-

etch replicas, appeared to have a bulbous end or "head"

(Fig. 45). Most viruses in more advanced polyhedra revealed

no such structures at either end.

Once the number of small inclusions in any one nucleus

reached a certain level, the rate at which individual in-

clusions grew decreased and they began to coalesce. This

resulted in the formation of many stick-like and club-shaped

small inclusions which measured 0.5 u in diameter and

averaged 20 to 4 u in length (Figs. 46 and 47). These in-

clusions coalesced further forming bundles with irregular

shapes (Fig. 48). These bundles then increased in size by

the addition of smaller inclusions or by fusing with each

other (Figs. 49-51). Eventually they condensed, forming

spindle-shaped inclusions which measured 2 to 4 u in diameter,

by 4 to 6 u in length. At this stage in polyhedra formation

a nucleus could contain over 100 small spindle-shaped inclu-

sions and the surface of these inclusions was rough. Figure

52 is representative of the polyhedral contents of an in-

fected nucleus at this stage of infection. The process
I










Figure 46.




Figure 47.











Figure 48.




Figure 49.











Figure 50.




Figure 51.


A scanning electron micrograph of a stick-like
inclusion typical of those seen in early stages
of polyhedral formation. 13,000 X.


A group of small stick-like and club-like inclu-
sions which have begun to coalesce. 6,000 X.










A bundle-shaped inclusion formed by the conden-
sation of several stick-like and club-like
inclusions. 12,000.


An ultra-thin section in which polyhedra have
begun to condense in the "ring zone" of the
nucleus. The larger inclusions at this stage
are bundle-shaped. 3.200.








A nucleus in a very late stage of infection.
Polyhedra development is well advanced and the
virogenic stroma has greatly atrophied. 4,500 X.


A section through a developing polyhedra in a
late stage of formation. Note that as the
virions are occluded the intimate and develop-
mental membranes condense tightly around the
nucleocapsid. 31,000 X.
















S.L


r-
pl


IrCm


is


f- /JIS.V


-- w. -






1.*'. *


!r ~ S

~r~cr








continued and the spindles coalesced further until a nucleus

at a late stage of infection would often contain only two or

three large large spindle- or ellipsoidal-shaped polyhedra

measuring 6 to 7 u in diameter, by 12 to 16 u in length

(Figs 53-56).

Scanning electron micrographs (Figs 52 and 53) and

fresh preparations examined under phase microscopy revealed

that most of the larger spindle and ellipsoidal forms had

rough surfaces. However, some spindles have been observed

in ultra-thin sections, thick epon-araldite sections, and in

scanning electron microscope preparations, which had very

smooth surfaces. This would suggest that either those forms

with the rugged surfaces were not completely condensed or the

possibility that they are non-viral in nature. The smooth-

and rough-surfaced forms behaved differently chemically.

Occasionally, inclusions developed in the cytoplasm of in-

fected cells (Fig. 56).

It is interesting to note that neither the crystalline

lattice nor the virus rods were ever observed in mature

polyhedra. Occasionally, a crystalline lattice was ob-

served in developing polyhedra in ultra-thin sections, and

linear arrays of protein molecules often appeared in develop-

ing polyhedra in freeze-etch replicas. In the later case,

the molecules within the polyhedra averaged 11.1 mu in

diameter, while those free in the nucleoplasm averaged

12.3 mu in diameter.










Figure 52.






Figure 53.










Figure 54.





Figure 55.









Figure 56.






Figure 57.


The polyhedral contents of a nucleus which was
osmotically shocked during an advanced stage
of inclusion formation. Note the fusiform shapes
and rugged surfaces on these inclusions. Scan-
ning electron micrograph, 2,400 X.


An advanced stage of inclusion formation in an
infected stomach cell nucleus. 3,500 X.









A portion of condensing inclusion. Note that as
the protein condenses the virions become less
distinct. No virions are discernible in the
densest area of the inclusion. 18,000 X.


Ellipsoidal inclusions at an advanced stage of
formation. Scanning electron micrograph, 2,500 X.


A stomach cell nucleus in a very late stage of
infection. The virogenic stroma has degenerated
and no virions are free in the nucleoplasm. Note
also the formation of inclusion in the cytoplasm
of this cell. 5,000 X.


An advanced inclusion in the nucleus of a cell in
the gastric caecae. the dense staining object
above the inclusion is a nucleolus. Inclusions
at this stage of form tion are very electron
dense. 7,500 X.






1 -


52


54


5 7A.4"."


e~ 8T"f.~~i&


E **.. ___


Oil


-~i.!










Figure 58. A spindle-shaped inclusion with a smooth surface.
Scanning electron micrograph, 10,000 X.


Figure 59. Membraneous lumellar organelles in the cytoplasm
of an infected gastric caecum cell. Note the
high level of ribosomal activity in this area.
8,100 X.





__ ___ __ ~ -.-....-...--.- -


Figure 60.






Figure 61.










Figure 62.


A segment of the nuclear membrane,seen in the
previous Figure, from an infected gastric cae-
cum cell. Note the'accumulations of granular
proteinaceous material on the outside of the
nuclear membrane. 40,000 X.


An infected stomach cell in which proteinaceous
granules have accumulated in the cytoplasm in
an area of heavy ribosome concentrations.
45,000 X.


A freeze-etch replica'of a fractured surface
through a membraneous organelle of the type
found in the cytoplasm of infected cells.
22,000 X.


Figure 63. A membraneous organelle similar to the one above.
Note the granular material associated with this
structure. 31,000 X4































~o.

a."


4


t i*": '
*- r ^


C -


- ~,.+


* -. ., -*. -. ---:. a


*O
'
'
r.








Membraneous Lamellar Organelles and Associated Proteins

In many infected cells, membraneous lamellar organelles

(Fig. 59) were found in the cytoplasm and appeared to be

associated with one or more proteins of viral nature which

accumulated in a granular form on the nuclear membrane and

in the cytoplasm (Figs. 60 and 61). These structures varied

in'size and shape, but most commonly they were spherical,

measuring from 1 to 2 u in diameter. In ultra-thin sections

the membranes of these structures were contorted, but in

freeze-etch replicas they appeared flat and stacked in parallel

layers (Figs. 62 and 63). Measurements made from freeze-

etch replicas indicated that the membranes ranged from 13 to

17 mu in thickness and the spaces separating them ranged from

26 to 48 mu. Globular structures, most likely protein in

nature, averaging 15 mu in diameter were often associated

with these membranes.


Mortality Studies

All inocula were diluted to contain 2 x 106 inclusions

per ml of inoculum.2 The results of all mortality trials are

presented in Table 3. The virus was most infective when lar-

vae were inoculated at 24 and 48 hours of age. When inocu-

lated at these times larval mortalities due to the virus were

36.5 per cent and 34.4 per cent respectively. The mortality

due to the virus decreased with the increasing age at which

the larvae were inoculated. The virus caused practically no


2 All shapes and sizes of inclusion bodies were counted.











O


0 4


0 0 -H0






-4 J 4 -.4

bo *,
>- H 01
04 04 O0
Cl U C


0)

1 4'
r-I C *.
0 04 $-



1 04 0r










H C)
I 4J 43
'd 0 -,-











0 4)J f
-H 4-'

0 0 0






H R



Ci
0 4 C











O 43
01
oC-


-' H. C

V 0 0
H -- 0


rl Co












en 0
,-'


0
H

l
H q













N me N
'V 'Vr O






en en en


0',

o



H to







n en H C Nt-


O LO N en en
o 9- ro NO

O H- C Ao ')
en o Co x Len


rie













c- CO
N '


CO


en, e












N H
Hl


r-I



CO

I r1


ro




1- 1;


Go Co













N Op
r- o)


01
C)
P)





01
0)
0 4i

C)






0 4J
o Cl





0I


CO
o i
4
4" rq




C 0
EC 0)
> 0

o- o,








larval mortality when larvae were inoculated at 96 hours of

age (third instar) or older.


Transovarial Studies

The results of Transovarial test #1 are presented in

Table 4. Fl larvae, whose parents had been exposed to the

virus as larvae, showed negligible differences in mortality

rates when compared with controls. However, out of 200 Fl

larvae reared, 4 developed patent infections.

The results of Transovarial test #2 are presented in

Table 5. These results indicate that the virus can cause

pupal mortality, but give no evidence in favor of transovarial

transmission of the virus.


Chemical Behavior of Inclusion Bodies

All rough-edged inclusions dissociated immediately on

being exposed to 1 N and 0.1 NaOH or HC1. In 0.01 N NaOH or

HCI the dissociation took place within 5 to 10 seconds. In

0.001 N NaOH the inclusions expanded and elongated initially,

and eventually dissociated after a period of 5-6 minutes. In

glacial acetic acid these inclusions elongated, lost their

refractile properties, and became more fusiform over a period

of. 30-40 seconds, but had not dissociated after 10 minutes.















Table 4.--Transovarial test 1: Mortality rates for F1
larvae reared from eggs collecLed from survivors
of inoculated and control larvaea'


Trial per cent
mortalities


Average
Mortalities


Control per cent
mortalities


10.3 10.0


0Average mortalities for the parent generation where
as follows: 24 hours, 45.6 per cent; 48 hours, 38.6 per
cent; 72 hours, 12.8 per cent; 96 hours, 9.8 per cent.

Lanvae per trial-200.

cOnly 4 patent infections were collected from these
trials.


Trial
No.


_~ C_ ~~ ~~__~_ ____ ___I









65







4,C)4J 0000 0000 0000 0000

'000
'O 0 0
o o






a
ra

41 -40 0 0 0-.0-0 i- O4 0 0 0 -4



H
) I



S 00






a0 C) 044


E-oa *4 OO HHo Heo ooH





0 m >, 0 o o . -


0 0 0



























00
0 0 o
H
0) OC)


rd ) 0 r,') rn c r '- D co i r r -J Co ) N

p N C) H m 0 o m co .-. r. r. . CO. I.. D . C)
0 a) r r-I -H H H H H-1 -l H H- H-H H- -
12, 0 N





E -1 0 0 0 0 -
0'0






4 H









H E- 0 0 0 0 0
> Q





(Td 4J
C) 3 co
C) rl 0













rI > u

El 1 3
iNE~. -

4.) C)
C) I




H )C ) )C
C)'E~U4 ~ 4i~~4J ~ U













DISCUSSION


In general, the symptoms of this nuclear polyhedrosis

disease were similar to those found in other insects where

the midgut epithelium was the site of infection (Aizawa, 1963).

The disease syndrome included a loss of appetite, sluggishness,

distension of the midgut, and the eventual development of a

chalky white appearance throughout the entire midgut epithe-

lium, including the cardia, gastric caecae, and the stomach.

This chalky white appearance was due to the hypertrophy of

infected nuclei packed with virus inclusion bodies. The inte-

gument rarely became discolored and larvae never lysed, prob-

ably because the epidermal tissues of the integument and the

fat body never developed patent infections. Since mosquito

larvae breed in a liquid environment, it could not be deter-

mined if unusual discharges were egested from the mouth or

anus; however, inclusion bodies were frequently present in

the feces of infected larvae. These inclusion bodies ap-

parently came from cells which were disrupted or sloughed off

during the course of the disease and in most cases were

probably carried through the gut, outside the peritrophic mem-

brane. When breeding in a natural habitat, this egestion of

inclusion bodies into the environment is probably very im-

portant in the dissemination of the virus throughout natural








populations of mosquito larvae. Diseased larvae frequently

discharge infective material in their feces for several days

before they die.

The direct cause of larval death is probably due to a

lack of sufficient nutrients to maintain life. After the cells

of the midgut epithelium have become infected, they undoubtedly

lose the digestive and absorptive properties of normal cells.

Food reserves are used, and the eventual atrophy of the fat

body and muscle tissue indicates that these tissues may be

either partially reabsorbed or that the diseased larvae is

incapable of acquiring enough nutrients to maintain them. In

later stages of infection, nuclei of the muscle cells fre-

quently hypertrophy, possibly as a result of a reabsorption

process. The fact that larvae which were infected in the late

third- or early fourth-instar, which already had a well-devel-

oped musculature and fat body tissues, did not die for several

days, even though the entire midgut epithelium was heavily

infected, indicated that the infection of this tissue alone

is not the direct cause of death.

With regard to time, the development of this disease,

with the observable multiplication of nucleoli and the hyper-

trophication of nuclei within 24-36 hours after infection, is

typical for NPV infections of the gut (Benz, 1963). The

destruction of host chromatin and the eventual development of

a nucleocapsid-producing, feulgen-positive virogenic stroma

in the center of the nucleus, and the eventual formation of

polyhedra in the "ring zone" agreed with the progression of

NPV diseases as described by Xeros (1955, 1956).








The occurrence of areas, within the stomach of infected

larvae, in which infected cells appeared to be proliferating

may be a result of a combination of the viral infection plus

the normal process of stomach cell regeneration and the pro-

duction of imaginal midgut cells. Richins (1945) in a study of

the development of the midgut in the larvae of Aedes dorsalis

(Meigen) stated that the regenerative cells of the stomach

are derived from epithelial cells in the anterior region of the

stomach, and that they dedifferentiate and undergo mitotic

divisions as they move posteriorly. The same process of regener-

ation most likely occurs in A. triseriatus,and it is possible

that if infection takes place in an area of mitotic divisions

the infected cells may give the impression of a proliferation

of diseased cells.

The actual process by which viruses attain entry into the

cells in which they replicate has not been elucidated (Smith,

1967; Vago and Bergoin, 1968). Leutenegger (1967) suggested

ingestion and phagocytosis of virus particles in the case of

Sericesthis iridescent virus. In the NPVs, the initial entry

into the host is usually from ingestion of virus-contaminated

food, but the process by which virions actually penetrate the

gut and enter other tissues is obscure. Aizawa (1962) and

Stairs (1968) state that polyhedra disappear from the gut of

B. mori within 20 minutes after ingestion and that polyhedra

were never found in the feces. Harrap and Robertson (1968)

found newly formed virus in the columnar cells of the midgut

with the NPV of A. urticae and suggested this was an important







source for the infection of other susceptible tissues, but

they did not explain how the virus originally entered the

gut cells. Kislev et al. (1969), studying the NPV attacking

the blood cells of the Egyptian cottonworm S. littoralis,

suggested that once visions or polyhedra were released from

infected cells into the hemolymph, they were phagocytized by

plasmatocytoids. In the case of mosquito larvae, the mode

of infection presents several additional problems. Clements

(1963) stated that dye studies indicated that ingested food,

enclosed in the peritrophic membrane, passes completely

through the alimentary canal of fourth-instar A. aegypti

larvae in 20-25 minutes at normal temperatures. There is no

reason to doubt that ingested material passes down the gut at

the same rate in A. triseriatus. It is conceivable that a

significant number of virions could rapidly be released

from polyhedra in the mildly alkaline conditions of the

mosquito gut, but it is difficult to imagine that these rods,

with a diameter ranging from 40 to 80 mu, are able to pass

through the peritrophic membrane, which, according to Dehn

(1933), retains particles larger than 25 angstroms.1

Examination of the midgut morphology of mosquito larvae,

and a study of the formation of the peritrophic membrane,

suggests two possible modes by which virions could come in

direct contact with midgut cells. The first case involves

moulting. According to Immes (1907), the peritrophic membrane


1. This figure is for insects in general. No figures
were available for the peritrophic membrane of mosquito larvae.








is shed through the anus during moulting. In this case the

peritrophic membrane must become detached from the cardial

cells which secrete it, and any virions which escaped from

the membrane as it passed posteriorly would remain in the

lumen of the gut. Conceivably, these virions would be in

direct contact with the midgut cells. The second possibility

involves the "penetration" of virions through areas where the

peritrophic membrane is secreted. Jones (1960) and Wiggles-

worth (1930) demonstrated that cardial cells secrete the pre-

cursor material of the peritrophic membrane, which is "rolled"

out and moved posteriorly by the anterior-posterior contrac-

tions of the oesophageal invagination. It seems possible

that virions could be drawn into this area by the motions of

zhe oesophageal invagination and would therefore come into

direct contact with the developing peritrophic membrane.

The current study presents limited evidence that this

latter situation may actually occur. The virions which are

shown in Figure 13 have accumulated along the developing

peritrophic membrane, but it is not clear whether the rods

have actually attached at this point. It must be realized

that these virions are not from polyhedra which have been

ingested recently, but are from midgut nuclei which have

broken down in the infected larva. This is apparent because

the virions are on the side of the peritrophic membrane

adjacent to the gut cells. The importance of such an accumu-

lation is that these virions may very well have an affinity

(or receptor) to components of the incompletely formed









peritrophic membrane in this area of the cardia. This hypotih

sis is further supported by two additional observations. Fir

in most cases the first cells to become infected were those

at the posterior end of the cardia, and, second, similar accur

ulations of virions were never observed further down the gut

where the peritrophic membrane had been completely formed.

Some workers have suggested that the "naked" viral genome

may be released in the gut and travel through the peritrophic

membrane to its site of infection. This seems unreasonable

because of the wide variety of enzymes present in the gut

which would most likely destroy the viral nucleic acid.

Once the midgut cells became infected, the process and

rate of the spread of the infection were dependent upon the

amount of infective material discharged into the lumen of the

gut (between the microvilli and the peritrophic membrane)

and the peristaltic and anti-peristaltic waves along the mid-

gut. Jones (1960) studied the rhythmical activities of the

midgut of Anopheles guadrimaculatus (Weidemann) larvae and

found that there were frequent peristaltic waves which were

always followed by anti-peristaltic waves. He stated that

these contractions served to move digested materials through-

out the gut to be absorbed by specific cells. In the present

study similar contractions were observed in the midgut of

A. triseriatus. In infected larvae these contractions also

resulted in a distribution of virions and inclusion bodies

throughout the entire midgut, thereby exposing all midgut

cells to virus. Host certainly this is the major mode of







disseminating infection throughout the gut. Although virions

have been seen "attached" to microvilli (Fig. 16), it can not

be stated emphatically that these virions were in the process

of infecting cells.

The increase in the number of nucleoli, typical of NPV
Pb
infections, was most likely responsible for the large increase

in the number of ribosomes found in the cytoplasm of infected

cells. This large increase in ribosomes also indicates that

most, if not all, viral proteins are synthesized in the cyto-

plasm and move into the nucleus. Protein synthesis in the

cytoplasm of cells infected with NPVs has been confirmed

histochemically by Benz (1960) and by Morris (1966, 1968)

among others.

The parallel aggregations of nucleocapsids which occur in

some nuclei are probably the result of temporary physiochemical

conditions within the nucleus; however, the possibility exists

that these aggregations may occur frequently but only at

certain stages of assembly and in localized areas of the

nucleus. The fact that holes are occasionally seen in the

center of cross-sections of newly formed nucleocapsids (Fig.

30) indicates that the structure of the nucleocapsis may be

very similar to that of Tobacco Mos&ic Virus as illustrated

by Caspar (1965). Harrap (1970) reported such holes as untra-

thin sections of P. dispar NPV, and Krieg (1961) indicated a

hollow core, the equivalent of this hole, in his NPV model.

The reason the hole is not seen in most cases is most likely

due to oblique sectioning of the nucleocapsids, or that the







nucleocapsids may be in a more condensed state. The source

of'the material from which the outer and intimate membranes

are derived was not identified, although it appears that the

internal membrane of the nuclear envelope may play some roll

in the formation of this material.2 The data presented in

this study indicate that the virus undergoes a process of

self-assembly typical of viruses in general (Caspar, 1965).

The coalescing process described in this study by which

inclusion bodies are formed is very different from the develop-

ment of most other polyhedra, such as those described by

Bergold (1963), and Summers and Arnott(1969). These reports

indicated that polyhedra grow individually by the simul-

taneous condensation of virions and inclusion body protein.

The actual time at which the coalescing process begins indi-

cates that this process may be directly under the influence

of the viral genome. This brings up the possibility that the

inclusion body protein may have allosteric properties, which

have been discussed by Monod et al. (1965). The fact that the

crystalline lattice of the inclusions was only occasionally

resolved indicates that the protein may not be arranged in a

face-centered cubic lattice (an orientation in which special

points of attraction on the protein molecules prevent them

from achieving their closest possible packing arrangement) as

described by Bergold (1963). It may also indicate the protein

molecules could be smaller than those characteristic of typ-

ical polyhedral inclusions. Similarly, Xeros (1966) never


2. See Appendix 3 for a discussion on the terminology
used to describe the morpology of nuclear polyhedrosis viruses.








observed a lattice structure in the inclusions of the NPV

attacking T. paludosa.

On the basis of the fusiform shape of the inclusion

bodies described in this study, this NPV must be considered

different from all other known NPVs described by Bergold

(1963), although it does appear to be somewhat similar to

the biconvex polyhedra described from the NPV of T. paludosa

by Xeros (1966). Clark et al. (1969) originally described

the polyhedra from A. sollictans as being cuboidal and ranging

in size from 0.1 u to slightly over 1.0 u in diameter. This

description was based on measurements made from electron micro-

graphs and the authors were obviously measuring incompletely

developed inclusion bodies. It is doubtful that the discre-

pancy in size and shape differences of the polyhedra was a

result of their description from a different host. However,

they described the virus rods as averaging 75 x 250 mu, and

the average measurements reported in this study were 63 mu x

200 mu. These differences may be a result of replication in

a different host, or to some extent, may be dependent upon

the time at which the rods were measured during inclusion

formation.

Although the inclusion body shape is different than those

of other NPVs, it is similar to the inclusion bodies of the

Vagoiavirus (Insect poxviruses) described by Vago (1963),

Weiser (1966), and Weiser and Vago (1966). In these reports,

the virus develops in the cytoplasm of fat body cells. The

spindle-shaped inclusions described in the cytoplasm of the

fat body cells of C. murinana infected with a- NPV are also








similar to the inclusions observed in this study (Huger and

Krieg, 1969). However, they differ greatly in size and

chemical properties and they contained no virions. The rela-

tionship between these various inclusion bodies and their

formation is not clear, but it appears as if there may be a

common shape to the protein molecules which make up the inclu-

sion bodies (Chun, personal communication).3

The smoothsurfaced, spindle-shaped inclusions which

occasionally developed in the cytoplasm were not purified

and it is not known whether they contained any infective

material, but it is doubtful that they did, as virions were

never observed in the cytoplasm.

The measurements of the virions reported in this study

are valid only for non-occluded virions and virions in early

inclusions. That is due to the fact that the virus undergoes

an unusual significant decrease in diameter, and to a lesser

extent a decrease in length, as the coalescing process takes

place. Figure 64 presents a schematic representation of

virions before and immediately after occlusion. After a cer-

tain point, only a few rods could be discerned within the

developing inclusions, and in fully developed fusiform inclu-

sions, the electron density of the inclusion was so great that

virions were never observed. This was probably due to the

fact that the virions and the inclusion body were of a uniform

electron density. It may be suggested that as the inclusion



3. Dr. Paul Chun, Prof..ssor of Biochemistry, College
of Medicine, University of Florida.






-80--


I
Jil
l i .'.:





. .
K !"i1 180 230
' '. : i)i H



1(..~ I

; V

V
*^ :< i


Outer
Membrane


Nucleocapsid


Intimate
Membrane


Non-occluded


Figure 64. A schematic illustration of completely
developed non-occluded and occluded
virions. Measurements in mu.


Occluded








condenses the virions move to a specific position within the

inclusion body; however, ultra-thin sections provided no evi-

dence for this and the process by which virions are observed

to be released from inclusion tends to support the former view.

No evidence was found for an inclusion body membrane sur-

rounding the final shape of the inclusion as described by Smith

(1967).

The fact that few, if any, rods are observed in nuclei

in which the fusiform inclusions were in a very advanced state

indicates that only one cycle of replication takes place within

a single nucleus, except perhaps in cases of multiple infec-

tions.

Proteinaceous fibers such as those reported by Summers

and Arnott (1969) in the cytoplasm and nucleoplasm of cells

infected with NPVs were not observed in this study, although

the accumulation of granular protein material on the external

surface of the nuclear envelope may be a related phenomena.

The function of the membraneous lamellar organelles seen

in the cytoplasm of infected cells may be involved in the pro-

duction of viral proteins although their possible role is

obscure. Freeze-etch replicas seemed to indicate that in-

clusion body protein may have some association with these

organelles. Their origin is unclear, but it is possible that

they are altered mitochondria. Bertram and B ird (1961) pre-

sented some evidence for this, except their observations were

based on similar organelles located in the midgut epithelium

of healthy adult A. aegypti females.








The reported mortality rates of 36.5 per cent and 34.4

per cent for larvae inoculated at 24 and 48 hours, respectively,

are greater than those of other mosquito viruses. Mortality

rates may be even higher for larvae which ingest virions or

inclusions immediately after hatching.

Results of transovarial tests were mostly negative and

the four patent infections which occurred in the first test

may have been the result of transovum transmissions. The fact

that the majority of the midgut tissue is discharged into the

gut lumen during pupation may explain this. If a larva devel-

oped a late infection, and during pupation shed the infected

midgut before the disease had progressed significantly, it is

very possible the adult would become contaminated with infec-

tive material on eclosion. This material if present on an

adult male could be mechanically transferred to the female

during copulation. Females contaminated on eclosion or during

copulation may in turn mechanically contaminate eggs during

oviposition.





































APPENDICES













Appendix 1. Staining Procedures for Light Microscopy



Heidenhain's Hematoxylvn

1. Remove paraffin with xylene.

2. Hydrate to distilled water through a descending

ethanol series.

3. Premordant in 2.5 per cent iron alum for 24-48 hours.

4. Stain overnight in 0.25 per cent Heidenhain's hema-

toxylin.

5. Rinse in running tap water for 5 minutes.

6. Differentiate in 2.5 per cent iron alum.

7. Rinse briefly in tap water containing a few drops

of concentrated ammonium hydroxide.

8. Rinse in slowly running tap water for 30 minutes.

9. Dehydrate to 70 per cent ethanol.

10. Stain for 2 minutes in 0.5 per cent eosin.

11. Dehydrate rapidly to absolute ethanol, clear in

xylene, and mount.

Feulgen Reaction

1. Remove paraffin with xylene.

2. Hydrate to distilled water through a descending

ethanol series.



1. Humanson, G. L., 1967.








3. Rinse for 2 minutes at room temperature in N HC1.

4. Iydrolyze at 600C in N HC1 for 8 minutes.

5. Rinse briefly in distilled water.

6. Stain in Schiff's reagent for 2 hours in total

darkness.

7. Transfer quickly to bleaching solution, 3 changes

for 2 minutes each.

8. Wash in running tap water for 15 minutes

9. Rinse briefly in distilled water.

10. Counterstain in 0.05 per cent fast green for

1 minute.

11. Dehydrate rapidly to absolute ethanol, clear in

xylene, and mount.

EHar's Stain for Polyhedra2

1. Remove paraffin with xylene.

2. Hydrate to distilled water through a descending

ethanol series.

3. 50 percent acetic acid for 5 minutes.

4. Rinse in distilled water for 2 minutes.

5. Azocarmine (Solution 1) for 15 minutes.

6. Rinse in distilled water for 5 seconds.

7. Aniline, 1 per cent in 95 per cent ethanol for 30

seconds.

8. Rinse in distilled water for 5 seconds.

9. Counterstain (Solution 2) for 15 minutes.



2. Harn, J. J., 1966.





82


10. Rinse and dehydrate in absolute ethanol, 2 changes,

30 seconds each.

11. Clear in xylene and mount.



Solution 1. Dissolve 0.3 gm of azocarmine G in 300 ml

of glacial acetic acid. Filter before use.



Solution 2. Dissolve in 300 ml of distilled water:

3.0 gm phosphotungstic acid, 0.3 gm aniline blue (water

soluable), 1.5 orange G, 0.6 gm fast green FCF.














Appendix 2. Fixation and Embedding Schedule for Electron
Microscopy



1. 3 per cent gluteraldehyde 2 to 3 hours at room

temperature.

2. Rinse in 0.1 M phosphate buffer overnight at 40C.

3. 1 per cent osmium tetroxide 2 hours at room temper-

a-ure.

4. 0.1 A phosphate buffer 15 minutes.

5. Dehydrate in ascending alcohol series 15 minutes

per step.

6. Propylene oxide, 2 changes 1 hour each.

7. Epon-araldite and propylene oxide (1:2) 2 hours.

8. Epon-araldite and propylene oxide (2:1) overnight

at 4C.

9. Pure epon-araldite 2 hours at room temperature.

10. Embed in pure epon-araldite.












Appendix 3. On the rTerminology Applied to the Morphology
and Anatomy of Nuclear Polyhedrosis and
Granulosis viruses



Bergold (1963), Smith (1967), and Summers and Arnott

(1969) state that the structure of the nuclear polyhedrosis

virus (NPV) rods and granulosis virus (GV) rods are the

same. Bergold (1963) describes these virus rods as con-

sisting of a dense, central, solid core surrounded by two

membranes; and intimate membrane which surrounds the dense

central core, and a developmental membrane which surrounds

the intimate membrane. These virus rods are then occluded

in a protein matrix known as an inclusion body. The major

difference between these two types of viruses lies in the

fact that the GVs are occluded in protein inclusions known

as capsules, one virus rod per capsule. However, the NPVs

have many virus rods, either singly or in bundles, occluded

in each large protein inclusion and this whole structure is

known as a polyhedra. The terminology used throughout the

insect virus literature to describe the developmental stages

and the resultant morphological or structural characteristics

of the GV and NPV rods is not constant, and therefore,

frequently perplexing.

Bergold (1950, 1952) first used the terms intimate

membrane and developmental membrane in proposing a life cycle










for the NPVs based on the morphological units of whole and

degraded virus rods he observed in negatively stained pre-

parations. He stated that in infected nuclei the virus

began as spheres on the developmental membrane and grew

within this membrane to a V-shaped form and finally to a

rod-shaped stage, by which time it had also acquired an

intimate membrane. Smith (1955) studying the NPV of T.

paludosa found there was a tendency for the particles to

have double membranes. He suggested that there was an

intimate membrane which, although not visible in ultra-thin

sections, held the material of the dense viral rod together.

Rods purified by centrifugation showed a definite outer

envelope or membrane and he stated that in sections this

membrane stands out some distance from the rod. He sug-

gested that there may be an inner intimate membrane closely

approximated to the rod itself. Xeros (1956) summarized his

work on the formation of NPVs in Lepidoptera and Hymenoptera

and stated that rods were released from vesticles and sur-

rounded by capsule membranes which secreted a capsule protein

around the rods. Xeros (1966) in a study of the NPV of T.

paludosa referred again to the outer membrane as a capsule

or theta (6) membrane. Bird (1957) referred to the outer

membrane observed in ultra-thin sections of the NPVs of D.

hercvniae and N. oratti banksianae as the developmental

membrane but did not mention observing an intimate membrane.








Day et al. (1958) studying ultra-thin sections of the NPV of

P. amilicornis used only the term membranes to refer to the

structures enclosing rods.

Krieg (1961) noticed a striated structure on the

intimate membrane, and discs with a hole in the center in

negatively stained preparations of NPV. On the basis of

these structures he proposed a model for NPV rods, which,

consisted of stacked discs tightly surrounded by an inti-

mate membrane, outside of which there was a space, followed

by developmental membrane. Bergold (1963) presented sche-

matic illustrations of the internal structure of the rods

of the NVs of V. mori and L. frugiperda as observed in

ultra-thin sections of polyhedra. In the B. mori illus-

tration the rod consists of a dense central core 38 mu in

diameter. From the edge of this core outwards, follow:

(a) a space 6 mu in width, (b) an intimate membrane 4 mu

thick, (c) another space 6 mu thick, and finally, (d) a

developmental membrane 7.5 mu thick. These rods are

occluded singly in polyhedra. The illustration for L.

fruginerda is similar, only in this case four rods sur-

rounded by individual intimate membranes surrounded together

by a common developmental membrane, forming a bundle of

virus rods. In polyhedra these rods are occluded in bundles.

Harrap and Juniper (1966) in negatively stained

preparations of the rods of the NPV of A. urticae refer to

Bergold's equivalent of a developmental membrane as an outer

membrane. They also noted an inner membrane which had a










regular repeating striated structure on its surface. In

a study of ultra-thin sections of the same virus, Harrap

and Robertson (1968) mention the outer membrane, but no

inner or intimate membrane. They use the term nucloocapsid

synonymously with virus rod. Harrap (1970) suggested the

inner membrane of the NPV of P. dispar, as seen in negatively

stained preparations, is probably the capsid.

Kozlov and Alexeenko (1967) studying negatively

stained preparations of D. mori NPV rods observed develop-

mental membranes and intimate membranes which showed regular

striated arrays of caposomeres. They proposed a model for

the virus rods of this NPV which contained a double layered

intimate membrane, surrounded by a developmental membrane.

Adams et al. (1968) studying ultra-thin sections of the NPV

of C. picta referred only to double membranes enclosing

virus rods to form rod packets, but they were no more

specific than this. However, their published electron

micrographs indicated that double membrane referred on3y

to the devclopmrental membrane of Bergold (1963).

Arnott and Smith (1968) in a study of the development

of a GV in Plodia intcrpunctellJa (I!u:bner) referred to naked

virus rods which they explained as virus rods lacking both

the ntimcate and developmental membrane.

I!im,.r,.o et al. (1969) referred to the outer membrane

of "intaLct parL.icl ; -" of the NI'V of B. nori. observed in

ultr'a-thin :("l.tions as "being doc.ble ayrued. 'J'hiIs icbran










surrounded what they called slender particles. Inner

membranes existed but could not be seen in ultra-thin sec-

tions of intact particles surrounded by an outer membrane.

However, they stated that inner membranes were easily se-n

in negatively stained preparations and had a regular repeat-

ing striated structure. Krieg and Huger (1969) studying

several NPVs in ultra-thin sections referred to naked virus

rods as having intimate membranes but not developmental

membranes which they obtained later. Rods devoid of any

membranes were referred to as virus threads which they said

were composed of helical DNA and protein.

In an attempt to clarify some of the above

descriptions, the following general comment is made. In

examining ultra-thin sections of nuclei infected with NPVs

and cells infected with GV, most workers observed an outer

or developmental membrane, which in some cases was double

layered. Some workers (Bergold, 1963; Arnott and Smith

1968) observed an intimate membrane while others either

didn't observe it or did not point it out specifically

(Harrap and Robertson, 1966; Adams et al. 1968; Bird, 1957;

Day et al. 1958; Xeros, 1956). However, in negatively

stained preparations the so-called (inner) intimate membrane

was seen by all workers, both empty and apparently containing

nuclear material, and it usually had a striated surface.

After e:m-ring the data presented in these papers

it becomes apparent that what some workers call the intimate




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