THE CYTOPATHOLOGY OF A NUCLEAR POLYHEDROSIS
VIRUS IN Aedes triseriatus (SAY)
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
TABLE OF CONTENTS
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
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
1. Staining Procedures for Light
Microscopy ................ ............... 80
TABLE OF CONTENTS-Continued
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
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
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
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
LIST OF FIGURES-Continued
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
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
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)
Brian Anthony Federici
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-
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
The results of transovarial studies were negative
although there was some evidence for trans-ovum traninmin-sion.
x i ; i.
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
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
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.
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
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
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
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
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
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
-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.
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
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
were examined .ith a ambridge Steroscan electron .-.icrcsco. e
at accelerating- voltages -- 5e an lC :V.
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, ...
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.
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
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
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.
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.
- 4 J
C) ) -H
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-
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
U- 4-) 0 -
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characteristic of tumor-like growths (Fig. 8). Just how
frequently this occurred was not determined, but it seemed
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.
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.
* '4~. i
4 '- -
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,
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.
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.
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.
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
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.
The "cords" of a well-developed
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.
I '. p l,
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
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
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.
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.
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
35.3 20.5 7.5 48.2 38.2 5.0
Table 2.-Dimensions of non-occluded and occluded whole virus
Whole Virus Nucleocapsid Whole Virus Nucleocapsid
Diameter Diameter Length Length
Non-occluded 79.4 39.4 236.0 184.0
Occluded 63.6 38.5 200.8 157.1
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.
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.
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
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
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.
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.
Vesicular envelopment' of-nfcleocapsids in
vesicles. 62,000 X.'
ji+ :LrS~ .i
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
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).
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.
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.
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
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
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.
-- w. -
!r ~ S
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
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.
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.
E **.. ___
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.
__ ___ __ ~ -.-....-...--.- -
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.
A freeze-etch replica'of a fractured surface
through a membraneous organelle of the type
found in the cytoplasm of infected cells.
Figure 63. A membraneous organelle similar to the one above.
Note the granular material associated with this
structure. 31,000 X4
t i*": '
*- r ^
* -. ., -*. -. ---:. a
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.
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.
0 0 -H0
-4 J 4 -.4
>- H 01
04 04 O0
Cl U C
r-I C *.
0 04 $-
1 04 0r
I 4J 43
'd 0 -,-
0 4)J f
0 0 0
0 4 C
-' H. C
V 0 0
H -- 0
N me N
'V 'Vr O
en en en
n en H C Nt-
O LO N en en
o 9- ro NO
O H- C Ao ')
en o Co x Len
larval mortality when larvae were inoculated at 96 hours of
age (third instar) or older.
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
Control per cent
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
_~ C_ ~~ ~~__~_ ____ ___I
4,C)4J 0000 0000 0000 0000
'O 0 0
41 -40 0 0 0-.0-0 i- O4 0 0 0 -4
a0 C) 044
E-oa *4 OO HHo Heo ooH
0 m >, 0 o o . -
0 0 0
0 0 o
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 -
H E- 0 0 0 0 0
C) 3 co
C) rl 0
rI > u
El 1 3
H )C ) )C
C)'E~U4 ~ 4i~~4J ~ U
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
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)
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
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.
l i .'.:
K !"i1 180 230
' '. : i)i H
*^ :< i
Figure 64. A schematic illustration of completely
developed non-occluded and occluded
virions. Measurements in mu.
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
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-
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
Appendix 1. Staining Procedures for Light Microscopy
1. Remove paraffin with xylene.
2. Hydrate to distilled water through a descending
3. Premordant in 2.5 per cent iron alum for 24-48 hours.
4. Stain overnight in 0.25 per cent Heidenhain's hema-
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.
1. Remove paraffin with xylene.
2. Hydrate to distilled water through a descending
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
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
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
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
8. Rinse in distilled water for 5 seconds.
9. Counterstain (Solution 2) for 15 minutes.
2. Harn, J. J., 1966.
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
1. 3 per cent gluteraldehyde 2 to 3 hours at room
2. Rinse in 0.1 M phosphate buffer overnight at 40C.
3. 1 per cent osmium tetroxide 2 hours at room temper-
4. 0.1 A phosphate buffer 15 minutes.
5. Dehydrate in ascending alcohol series 15 minutes
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
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
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,
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