• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Review of literature
 Materials and methods
 Results and discussion
 Summary and conclusion
 Appendix
 Appendix 1
 Appendix 2
 Literature cited
 Biographical sketch
 Copyright














Title: The pathobiology of a mosquito iridescent virus in Aedes taeniorhynchus (Wiedemann)
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
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Permanent Link: http://ufdc.ufl.edu/UF00084185/00001
 Material Information
Title: The pathobiology of a mosquito iridescent virus in Aedes taeniorhynchus (Wiedemann)
Physical Description: 74 leaves : ill. ; 28 cm.
Language: English
Creator: Hall, Donald W
Publication Date: 1970
 Subjects
Subject: Mosquitoes   ( lcsh )
Virus diseases   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Includes bibliographical references (leaves 69-72).
Statement of Responsibility: by Donald William Hall.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00084185
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000441450
oclc - 37752871
notis - ACK2033

Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
    List of Figures
        Page v
    Abstract
        Page vi
        Page vii
    Introduction
        Page 1
        Page 2
        Page 3
    Review of literature
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
    Materials and methods
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Results and discussion
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
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        Page 49
        Page 50
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        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
    Summary and conclusion
        Page 62
        Page 63
        Page 64
    Appendix
        Page 65
    Appendix 1
        Page 66
        Page 67
    Appendix 2
        Page 68
    Literature cited
        Page 69
        Page 70
        Page 71
        Page 72
    Biographical sketch
        Page 73
        Page 74
    Copyright
        Copyright
Full Text









THE PATHOBIOLOGY OF A MOSQUITO IRIDESCENT

VIRUS IN Aedes taeniorhynchus (Wiedemann)















By
DONALD WILLIAM HALL















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











UNIVERSITY OF FLORIDA

1970














ACKNOWLEDGEMENTS


Gratitude is expressed to the members of the writer's committee:

Dr. F. S. Blanton, Dr. G. E. Gifford, Dr. D. H. Habeck, Dr. L. C. Kuitert,

and especially to Dr. R. E. Lowe under whose guidance this study was

initiated.

Appreciation is also expressed to the staff of the Insects Affecting

Man and Animals Research Laboratory, U. S. Department of Agriculture

for the use of their facilities during this study. The writer is

especially indebted to Mr. D. W. Anthony for his generous assistance with

the electron microscopic studies.

Finally, the writer would like to thank his wife, Diane, for her

help and patience throughout this study.















TABLE OF CONTENTS



Page

ACKNOWLEDGEMENTS .... . . . ... .. ii

LIST OF TABLES . . . . . . iv

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

ABSTRACT . . . . . vi

INTRODUCTION . . . . . 1

REVIEW OF LITERATURE . . . . . 4

MATERIALS AND METHODS . . .. . . 10

Infection and Rearing of Larvae . . . .. 10
Light and Electron Microscopic Methods . . .. 10
Transovarial Transmission Studies . . . 11
Purification of Virus ....... ........ .... 12
Preparation of Antiserum . . . . 13
Immunodiffusion . . . ...... 14
Physical Comparisons of RMIV and TMIV . . .. 16
Comparison of Iridescence of RMIV and TMIV . .. 16

RESULTS AND DISCUSSION ... . . ... 17

Incidence of MIV in Nature . . . . 17
Pathology of RMIV . . 17
Transovarial Transmission ... .. ... .. 42
Immunodiffusion . . . . 47
Physical Comparisons of RMIV and TMIV . .. .. 52

SUMMARY AND CONCLUSIONS . .. . . .. 62

APPENDIX 1 . . . . . 66

APPENDIX 2 . . . . . 68

LITERATURE CITED . . . ., 69


~_~ _C_ ~ ~rl~ ~ I _~(__ __~II















LIST OF TABLES


Table Page

1. Incidence of RMIV in A. taeniorhynchus larvae 18

2. Transovarial transmission of RMIV . . . 48

3. Side-to-side measurements of RMIV and TMIV from electron
micrographs of tissue sections .. . . .. 55


Siv














LIST OF FIGURES


Figure Page

1. Section of healthy A. taeniorhynchus larva . . 21

2. Section of infected A. taeniorhynchus larva showing
densely staining cytoplasmic inclusions in fat body and
tracheal epithelium .. . . . ... 23

3. Fat body cells with virus packed in paracrystalline arrays 25

4. Infected fat body and tracheal epithelium. Virus particles
not packed in arrays . . ........ 27

5. RMIV in epidermal cell . . . .. 30

6. Infected imaginal disc . . . . 32

7. Site of viral replication in a cell of the esophageal
invagination . . . .. . 36

8. Hemocyte with virus particles enclosed in vacuolar
structures . . . . . 39

9. Infected nerve . . . . . 41

10. Larval testis of A. taeniorhynchus surrounded by infected
fat body . . . . . . 44

11. Virus in nucleated sheath of larval ovary . .. 46

12. Serological comparison of RMIV and TMIV using concentric
pattern . . . . . 51

13. Serological comparison of RMIV and TMIV using the 4 well
square pattern . . . .. .. .. 51

14. Comparison of RMIV and TMIV in sucrose density gradients 54

15. Tubular structures in imaginal disc tissue infected with
RMIV . . . . . .. 58

16. Purified pellet of RMIV . . . . 59

17. Purified pellet of TMIV . ....... 60









Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy



THE PATHOBIOLOGY OF A MOSQUITO IRIDESCENT
VIRUS IN Aedes taeniorhynchus (Wiedemann)


by

Donald William Hall

August, 1970



Chairman: F. S. Blanton
Co-Chairman: R. E. Lowe
Major Department: Entomology


An investigation was initiated to study the pathology and biology of

the regular mosquito iridescent virus (RMIV) in the black salt marsh

mosquito, Aedes taeniorhynchus (Wiedemann). RMIV was capable of infecting

a variety of tissues within its host.' Cells of the fat body, tracheal

epithelium, imaginal discs, and epidermis were the primary sites of viral

replication. Extensive destruction of the fat body by this virus resulted

in the death of most infected mosquitoes before they reached the adult

stage. Other tissues which were involved to a lesser extent were hemocytes,

esophagus, nerve, muscle, and both larval and adult ovaries.

The transovarial transmission of RMIV was confirmed, and when

transovarial transmission occurred, either all or none of the progeny of

a given female were infected. The presence of virus in the ovaries was

taken as evidence that RMIV is transmitted within the egg.

Immunodiffusion experiments with alkaline degraded virus revealed the

presence of 4 antigens which were shared by RMIV and the turquoise mosquito









iridescent virus (TMIV). While purifying RMIV and TMIV, it was observed

that they sedimented at different rates in sucrose density gradients.

Subsequently, measurements from electron micrographs revealed that RMIV is

about 195 my and TMIV is about 160 my in diameter. When the 2 viruses

were subjected to equilibrium ultracentrifugation in cesium chloride,

RMIV banded at a lower level than TMIV indicating that it is slightly

more dense than TMIV. These comparisons of the serological and physical

characteristics of RMIV and TMIV indicated that they are closely related

but not identical.


.. vii














INTRODUCTION


Recently there has been an increasing awareness of the ecological

dangers associated with the use of persistent pesticides. Pressure has

been mounting from many conservation organizations and numerous concerned

individuals to phase out the use of these chemicals. One such group

of pesticides which has been under particular attack is the chlorinated

hydrocarbon group, of which l,l,l-trichloro-2,2-bis(p-chlorophenyl)

ethane (DDT) is the best-known member. Several states have already

taken steps to limit the use of DDT, and related compounds, because of

their effect on certain wild animal populations, primarily those high

in the food chain. Many of these chemicals undergo biological magni-

fication as they move through the food chain until they finally reach

concentrations which are apparently damaging or lethal to certain organisms.

It is impractical to discontinue the use of these pesticides without

first finding acceptable methods of pest control to take their place.

One possible alternative or supplementary measure to the use of chemicals

for insect control is the utilization of pathogens, parasites and predators.

An advantage of such biological controls is that many such agents are

relatively host specific in contrast to most insecticides which have a

broad spectrum of activity.

Mosquito control is one situation in which persistent insecticides

have been used extensively, and these chemicals have proven especially

effective when used as larvicides in areas which are flooded periodically.

This application appears to be potentially one of the most harmful from

1









an ecological standpoint. For this reason, biological control of

mosquitoes is a highly desirable objective.

Biological control of mosquitoes appears to be more feasible than

with many other insects due to the biology of mosquitoes. Pathogens

normally require from several days to a week to kill the host and many

insects do most of their damage during the immature stages. Consequently,

they would do considerable damage before being killed by pathogens.

In contrast, only adult mosquitoes are destructive as pests or vectors

of disease. Since most of the known pathogens of mosquitoes are capable

of killing the host during the larval stage, control could be accomplished

before the mosquito reaches the destructive stage.

Currently, the iridescent viruses of mosquitoes are being studied

for their potential as agents of biological control. If methods can be

found to increase the infection rates and circumvent the problem of

instability, these viruses may have greater potential for control.

Before any pathogen can be used effectively, a large amount of basic

information is needed concerning the biology of the pathogen and its

effect not only on the host, but also on other organisms. The purpose

of the present study was to gain information relative to the biology and

taxonomic relationships of 2 iridescent viruses of the black salt

marsh mosquito, Aedes taeniorhynchus (Wied.), with the goal that someday

they may be used for control of this species.

A. taeniorhynchus is the most important pest mosquito along most

of the southeast Atlantic coast as well as the entire coast of the Gulf

of Mexico. Large populations of this mosquito are responsible for

heavy financial losses in many coastal areas each year due to the loss

of tourist trade and damage to livestock. For this reason, there is a





3


desperate need for effective measures to control this pest, which are

not hazardous to the ecology of the tidal estuaries.













REVIEW OF LITERATURE


A cytoplasmic virus of the European crane fly, Tipula paludosa

(Meigen), was described by Xeros (1954) which resulted in an iridescent

blue color being imparted to larvae when viewed in direct sunlight.

Williams and Smith (1958) observed the same iridescence in purified

pellets of the virus and designated it the Tiputa iridescent virus (TIV).

Iridescent viruses imparting various colors of iridescence have since

been reported from a scarabaeid beetle larva, Sericesthis pruinosa

(Dalman), by Steinhaus and Leutenegger (1963); from a mosquito, A.

taeniorhynchus (Wied.), by Clark et al. (1965); from the rice stem borer,

Chilo suppressalia (Walker), by Fukaya and Nasu (1966); from a black fly,

SimuZiwn ornatwn Meigen, by Weiser (1968); from a sand fly, Culicoides

sp., by Chapman et al. (1968); and from a phantom midge, Corethrella

sp., by Chapman (1970).

The taxonomic status of the iridescent viruses is still uncertain

and at the present time they are generally designated by either the first

letter or the first 2 letters of the genus name of the host, followed

by the letters IV for iridescent virus (e.g., TIV for Tipula iridescent

virus). An exception to this convention is the abbreviation MIV for the

mosquito iridescent viruses.

Mosquito iridescent viruses have been described from 7 different

species of mosquitoes. The first was described from A. taeniorhynchus

collected at Vero Beach, Florida (Clark et al., 1965). This virus

exhibited a brownish-orange iridescence and has since been designated






5

the R (regular) MIV (Matta and Lowe, 1970). Weiser (1965) described

an iridescent virus from Aedea cantans (Meigen) and Aedes annulipes

(Meigen) in Czechoslovakia. Subsequently, Chapman et at. (1966) reported

iridescent viruses from Aedes fulvus pallens Ross, Aedes vexana (Meigen),

and Psorophora ferox (Humb.). A second iridescent virus was reported

from A. taeniorhynchus by Woodard and Chapman (1968) which produced

a blue-green iridescence and has been designated the T (turquoise)

MIV (Matta and Lowe, 1970). Recently, another iridescent virus has

been reported from Aedes stimulans (Walker) by Anderson (1970). The

relationships between the members of the mosquito iridescent virus group

have not been investigated.

Serological comparisons of Sericesthis iridescent virus (SIV) and TIV

have shown these viruses to be related but not identical (Day and Mercer,

1964; and Cunningham and Tinsley, 1968). Cunningham and Tinsley (1968)

reported that MIV does not cross react serologically with either SIV or

TIV..

Much of the information on the structure of the iridescent viruses

and their crystals is based on work with TIV and SIV. Williams and Smith

(1958) demonstrated the icosahedral shape of TIV by shadowing, in 2

directions differing by 60 in azimuth, virus particles which had

been freeze-dried, and observing the shape of the resulting shadows.

Thomas and Williams (1961) demonstrated by electron microscopy of

enzymatically digested virus particles that the outer shell of TIV is

protein and that the inner core is nucleoprotein.

Wrigley (1969) proposed a structure for SIV with 1,562 morphological

subunits and a triangulation number of 156. His proposed structure was

based on electron microscopy of negatively stained preparations which had








been previously treated with the nasal decongestant "Afrin" to reveal

surface structure.

It has been shown that the iridescent effect is the result of dif-

fraction of the visible light by crystalline arrays of the viruses (Williams

and Smith, 1957). Klug et al. (1959) have conducted crystallographic

studies with TIV and reported that the virions are packed in a face-centered

cubic array with an interparticle spacing which is nearly twice the

diameter of the freeze-dried particle and presumably consists of only

water. Mercer and Day (1965) have presented several lines of evidence

which suggest that in crystals of SIV, the virions are separated by a

diffuse surface coat which is invisible in the electron microscope.

The physicochemical properties of TIV, SIV, and CIV have been

extensively studied (Thomas, 1961; Bellett and Inman, 1967; Kawase and

Hukuhara, 1967; Glitz et al., 1968; and Kalmakoff and Tremaine, 1968),

and Bellett (1968) has reviewed the properties of these viruses. Matta

(1970) characterized RMIV, and reported a buoyant density of 1.354 g/cm3

an average diameter of 180 mp, and a sedimentation coefficient of 4,458.
9
He also reported a particle weight of 2.486 x 10 daltons of which approxi-

mately 16 per cent is DNA. Faust et al. (1968) reported values of 11.7

per cent and 10.5 per cent DNA for RMIV and TMIV respectively, but these

values appear to be too low since their virus was probably contaminated

with top component (Matta, 1970).

TIV and SIV appear to have wide host ranges experimentally. In

addition to T. paludosa, the original host of TIV, larvae of 7 species

of Diptera, 11 species of Lepidoptera, and 3 species of Coleoptera have

been infected with this virus in the laboratory (Smith et al., 1961).

SIV has been successfully transmitted to GalZeria mellonella (Linnaeus)








and Tenebrio monitor Linnaeus (Steinhaus and Leutenegger, 1963) in addition

to its original host, S. pruinosa. Malyuta and Aleksandrov (1969) have

induced high mutagenic activity in Drosophila melanogaster Meigen

by injection of TIV or MIV into adult males. It is not known whether

the viruses replicate in this host.

The range of insects which can be infected with mosquito iridescent

viruses has not been adequately studied. In preliminary tests, Woodard

and Chapman (1968) reported transmission of certain of the mosquito

iridescent viruses from their original hosts to other species of mosquitoes.

In their study, Aedes sollicitans (Walker) was infected readily by both

RMIV and TMIV, although patent MIV infections of this species have

never been found in the field. This is surprising in view of the fact

that it is commonly found in the same pools with infected A taeniorhynchus.

TIV, SIV, and CIV have been successfully inoculated into tissue

cultures. Bellett (1965a) has developed an in vitro assay for SIV in

Grace's Antheraea eucalypti cell line by counting virus infected cells

stained with fluorescent antibody. Syntheses of both viral protein and

viral DNA have been shown to take place in cytoplasmic foci of SIV

replication (Bellett, 1965b). Bellett and Mercer (1964) reported that

complete virions of SIV appeared to be released from A. eucalypti cells

by a budding process during which the virion acquired an envelope derived

from the cell membrane. The same type of mechanism has been described

for CIV (Hukahara and Hashimoto, 1967) and TIV (Younghusband and Lee,

1969). A lipid envelope is not essential for infectivity of SIV (Bellet,

1965b; Day and Mercer, 1964) or MIV (Matta, 1970).

Several studies have been conducted to determine the range of tissues

infected by iridescent viruses. Bird (1961) reported on the basis of









electron microscopic studies, that cells of the silk gland, fat body,

epidermis, tracheal epithelium, and muscles of T. paludosa and G. mellonella

were infected by TIV. The sites of infection by CIV in C. suppressalis

were determined by Mitsuhashi (1966) on the basis of the appearance

of iridescence in the tissues. In addition to the tissues listed

above for TIV, CIV was reported to infect the alimentary canal,

malpighian tubules, salivary gland, pericardial cells and subesophageal

gland cells. The iridescent virus from A. stimuZans was reported

to infect pericardial cells, oenocytes, epidermis, sarcolemma, fat

body, ovaries, testes, and imaginal discs (Anderson, 1970).

Woodard and Chapman (1968) studied the transmission of RMIV and

TMIV in A. taeniorhynchus. They reported an average transmission of

16 per cent for 68 serial passages, but that the rates varied greatly

between these passages as well as between replicates within a single

passage. Transmission was accomplished by exposing late first-instar

or early second-instar larvae to the virus. In the same study it

was observed that a small number of larvae from eggs laid by survivors

of infection tests were always infected. Subsequently, it was found

that larvae exposed to the virus in the late third-instar or early

fourth-instar did not develop patent infections, but about 20 per

cent of their progeny were infected. Linley and Nielsen (1968a)

confirmed these results and also suggested that when transovarial

transmission occurs either all or a very high proportion of the

progeny from an infected female are infected. In addition, they

were able to rear some patently infected larvae to the adult stage

and obtain eggs from them. They reported that neither the larvae

from these eggs, nor the next 2 succeeding generations developed patent






9


infections. On the basis of field experiments, Linley and Nielsen (1968b)

proposed an account of the natural history of MIV. They suggested that

MIV exists by a simple cycle in which transovarial transmission gives

rise to infected larvae which die in the fourth-instar. The cadavers

are fed upon by other fourth-instar larvae prior to pupation. Adults

which develop from these larvae then complete the cycle by passing the virus

on to their progeny.














MATERIALS AND METHODS


Infection and Rearing of Larvae

Twenty-four-hour-old A. taeniorhynchus larvae, from an established

laboratory colony, were infected with MIV by exposing them overnight in

small, plastic petri plates (60 x 15 mm) in a crude homogenate of infected

larvae. The homogenate was prepared by grinding 7 infected fourth-instar

larvae in 5 ml of 0.15 per cent NaC1 in a Ten Broeck tissue grinder. After

exposure to the virus, larvae were transferred to Plexiglas trays containing

approximately 6 liters of 0.15 per cent NaC1 plus 50 ml of hay infusion

and reared to the fourth-instar in a room maintained at 31C. High protein

hog supplement was added as needed for food. Air was bubbled through

the rearing medium to prevent the formation of scum on the water surface

which would have suffocated the larvae. Fourth-instar larvae were screened

for patent infection in black photographic trays. Infected larvae were

stored in a small amount of saline in an ultralow temperature freezer

at -65C until needed.

Light and Electron Microscopic Methods

Specimens to be used for histological examination by light microscopy

were fixed for 4 hours in Carnoy's fixative (Humason, 1967), dehydrated

through ascending concentrations of alcohol, infiltrated successively


1. Purina S.E. hog supplement 40% (H). Ralston Purina Company, St.
Louis, Missouri.








2
with tertiary butanol and butanol Paraplast mixtures, and embedded

in Paraplast. Sections were cut at a thickness of 6 P and stained with

Heidenhain's hematoxylin or by the Feulgen technique (Appendix 1). When

sections were to be stained by the Feulgen technique, it was necessary to

pre-treat the slides with an adhesive to prevent the loss of sections

during the acid hydrolysis part of the staining schedule. The slides

were first coated with a thin film of Haupt's adhesive (Johansen, 1940)

and then flooded with a 3 per cent solution of formalin. Tissue sections

were floated onto the formalin and dried overnight on a slide warmer.

Specimens for electron microscopy were cut into small pieces (approx-

imately 1 cubic mm) and fixed for 3 hours with 3 per cent glutaraldehyde

in 0.1 M phosphate buffer, at room temperature. The specimens were left

overnight in buffer; then they were post-fixed in 1 per cent osmium

tetroxide in 0.1 M phosphate buffer for 2 hours, dehydrated through

ascending concentrations of ethanol, and embedded in Epon 812 (Luft, 1961).

The complete fixation and embedding schedule is given in Appendix 2.

Ultrathin sections were cut with glass knives on a Sorvall MT-2 ultramicro-

tome and stained with saturated uranyl acetate, followed by lead citrate

(Venable and Coggeshall, 1965). Thick sections (4 V) were also cut and

examined by phase-contrast microscopy to aid in orientation. Thin sections

were examined and photographed with a Hitachi 125E electron microscope

using accelerating voltages of either 50 or 75 kV.

Transovarial Transmission Studies

For transovarial transmission studies, 100 third-instar A. taenio-

rhynchus larvae were placed in 50 ml beakers containing 50 infected cadavers


2. Scientific Products, Evanston, Illinois.









in 20 ml of 0.15 per cent NaC1 and allowed to feed overnight. Controls

were set up in the same manner except that these larvae were fed on

uninfected cadavers. After treatment, each group was placed in an enamel

pan with 1 liter of 0.15 per cent NaC1 and reared to the adult stage on

a diet of high protein hog supplement. Adult females were offered guinea

pigs as a source for a blood meal. Females which had been exposed to the

virus, and that had taken blood meals, were separated into individual,

numbered vials with screen tops. A gauze pad, soaked in hay infusion,

was placed in the bottom of each vial as a site for oviposition, and a

moistened cotton pad and a raisin were placed on top of each vial as a

food source. Control females were allowed to oviposit in a common con-

tainer. After oviposition, the females were immobilized and their ovaries

were removed, fixed for electron microscopy (Appendix 2), and held in 70

per cent ethanol. The eggs from each female were allowed to hatch, and

the F larvae were reared to the fourth-instar as before and screened for
2
infection. Ovaries from the females which produced infected progeny were

prepared for electron microscopy (Appendix 2) and examined for the presence

of virus.

Purification of Virus

Infected larvae were triturated using either 7 or 15 ml Ten Broeck

tissue grinders containing 0.055 M phosphate buffer. Thirty ml of homo-

genate was centrifuged at 1700 RPM in a clinical centrifuge for 5 minutes,

and the supernatent was strained through 2 layers of organdy to remove

large insect fragments. The supernatant was diluted so that the virus

from 50 larvae was suspended in 10 ml of buffer. The suspension was then

subjected to 2 alternate low and high speed, differential centrifugation

cycles (4,000 RPM for 10 minutes and 20,000 RPM for 30 minutes) using









a Spinco type 50 angle head rotor in a Beckman model L ultracentrifuge.

After each high speed cycle, the virus pellet was allowed to soften

overnight in fresh buffer before being resuspended. After the second

high speed cycle, the virus pellet was gently washed with buffer to remove

a thin pellicle of noniridescent material from the top of the pellet. The

virus was further purified by 2 cycles of sucrose density gradient centri-

fugation. Sucrose gradients were prepared by layering 0.9 ml volumes of 54,

44, 30, 16, and 6 per cent solutions consecutively into a 5 ml cellulose

nitrate tube and allowing the gradient to form overnight at 4C. From 0.3

to 0.5 ml of virus suspension was layered onto each gradient and centrifuged

at 15,000 RPM in a SW 39L rotor until the visible virus bands were near the

middle of the tube. RMIV was centrifuged for 10 minutes to achieve this while

TMIV required about 15 minutes. The virus bands were collected with an ISCO

model D density gradient fractionator, or by very carefully removing them

by hand with a syringe. After each sucrose density gradient cycle, the virus

was pelleted as before, washed with buffer, and resuspended in 0.055 M

phosphate buffer. Purified virus was stored at -70*C until needed. Virus

prepared in this manner was free of other particulate matter and serologically

pure at the level of sensitivity of the gel diffusion tests used. Matta (1969)

reported that RMIV prepared in essentially the same way exhibited a single

peak during sedimentation in a Beckman model E analytical centrifuge with

Schlieren optics.

Preparation of Antiserum

Antiserum to both RMIV and TMIV was produced by injecting the purified

viruses into rabbits. Two intramuscular injections, each containing approx-

imately 5 mg of virus in emulsion with Freund's complete adjuvant, were

given to each rabbit 1 month apart. These injections were followed 2 weeks








later by an intravenous injection containing 5 mg of virus in saline.

A blood sample was collected from the ear vein of each rabbit 2 weeks

after the last injection. The blood was allowed to clot for 2 hours at

room temperature and kept overnight at 4C to allow the clot to retract.

The serum was then collected, centrifuged to sediment erythrocytes, and

stored at -20C for immunodiffusion studies.

Immunodiffusion

Serological comparisons of RMIV and TMIV were made by double

diffusion in 0.75 per cent Difco purified agar, in a solution of 0.15 M

NaC1 and 0.05 per cent sodium azide. Gel diffusion tests were conducted

in 100 x 15 mm plastic petri plates containing 12 ml of agar per plate.

Two diffusion patterns were used in the present study. The first was

a 7 well pattern in which there were 6 peripheral wells (7 mm in diameter)

surrounding a center well (9.5 mm in diameter) at a distance of 10 mm.

The second was a 4 well square pattern with the adjacent wells (7 mm in

diameter) separated by a distance of 4 mm (Shandon Scientific Co., 1959).

The square pattern was designed to allow simultaneous comparison of 2

antigen-antibody systems and is particularly useful in demonstrating

antigens unique to each system. Virus used in most gel diffusion tests

had been partially purified by 2 differential centrifugation cycles.

Due to their large size, the iridescent viruses did not diffuse

readily in agar gels, and for this reason several different methods were

used to degrade them. Alkaline degradation was accomplished by treating

virus preparations with 0.05 N NaOH and also by dialysis (48 hours at

40C) against either 0.2 M 2-aminoethanol (pH 11), 0.2 M 2-aminoethanol

(pH 10.5), or against 0.1 M Tris (hydroxmethyl) aminomethane (pH 10.5).

Also, disruption of the viruses was attempted by treatment with a 1 per cent









solution of the detergent Leonil SA. Another method by which the problem

of diffusion was circumvented was by the use of highly concentrated, crude

homogenates of RMIV and TMIV infected larvae. These were prepared by

grinding 60 infected larvae in 5 ml of saline and sedimenting the larger

insect fragments and cellular debris in a clinical centrifuge. The result-

ant supernatent was used in subsequent tests without further treatment.

The concentration of virus in the antigen preparations was not

determined because of the impurities present*, however, each of 5 serial,

2 fold dilutions of the original preparations were tested against each

antiserum using the 7 hole concentric pattern. Also, each antiserum was

tested against 5 serial, 2 fold dilutions of a control homogenate consist-

ing of 60 uninfected larvae ground in 10 ml of saline. In addition,

controls were set up to test for nonspecific precipitates due to buffers

and normal rabbit serum.

Gel diffusion plates were incubated in a chamber with increased

humidity for 24 hours at 260C. Plates were observed by using a dark-field

illumination box of the type described by Crowle (1961). Plates to be

photographed were incubated for an additional 24 hours, either at 26C

or in the refrigerator, to enhance the formation of precipitin bands.

These plates were photographed with a Polaroid MP-3 camera using Polaroid

type 55 P/N film with dark-field illumination. Attempts were made with

some plates to dry the agar gels and stain the precipitin bands for

photography with either thiazine red R or crocein scarlet MOO (Crowle,

1961). Selected gel diffusion plates were treated with a 0.01 per cent

solution of cadmium chloride. With some antisera, this treatment has been

shown to develop precipitin bands which were previously invisible (Crowle,

1961).









Physical Comparisons of RMIV and TMIV

The relative sedimentation rates of RMIV and TMIV were compared in

sucrose density gradients. For this comparison a suspension of each

virus was layered on different density gradient tubes and a mixture of

the 2 viruses was layered on a third tube. The tubes were spun for 20

minutes at 12,000 RPM in a SW 39L rotor and examined for the number and

relative positions of bands. Similarly, the viruses were compared for

differences in buoyant density by equilibrium ultracentifugation in cesium

chloride. This was performed as described by Matta (1970).

Size comparisons of RMIV and TMIV were made from tissue sections.

Direct measurements were made from photographic negatives by the use of a

dissecting microscope with a calibrated ocular micrometer, and these were

corrected for the magnification of the electron microscope. The actual

electron microscope magnification was determined by photographing a grating

replica3 containing 54,800 lines per inch, at the same magnification step

at which the viruses were photographed.

Comparison of Iridescence of RMIV and TMIV

The iridescence produced by purified pellets of RMIV and TMIV was

compared visually after centrifuging purified virus suspensions for 15

minutes at 25,000 RPM in a SW 39L rotor. Comparisons were made after

centrifugation in solutions of increasing salinity from distilled water

to 0.16 M NaCl. Attempts were made to study the crystalline structure of

these pellets by electron microscopy, but difficulties were encountered

in infiltrating the pellets with plastic.



3. Ernest F. Fullam, Inc., P. 0. Box 444, Schenectady, New York














RESULTS AND DISCUSSION


Incidence of MIV in Nature

A group of A. taeniorhynchus larvae collected June 17, 1969, from

North Key, an island off the west coast of Florida, near Cedar Key, were

examined and 2 were found to be infected with RMIV. Three additional

collections were made during the summer from North Key and 1 from a

second island, Atsena Otie Key, to determine the incidence of infection.

The number of patently infected larvae in these collections is shown in

Table 1, and these results are comparable to those reported by Chapman

et al. (1966) from collections in Louisiana.

The location of these collections, in addition to those reported

previously (Clark et aZ., 1965; Chapman et aZ., 1966), suggests that RMIV

is present throughout the distribution range of A. taeniorhynchus. It

is of interest that TMIV has been reported only from Louisiana, and was

not observed in any of the collections from the west coast of Florida.

This indicates that TMIV may not be as prevalent as RMIV, although this

may simply reflect the lack of a concentrated search for MIV in Florida.

Pathology of RMIV

The range of tissues of A. taeniorhynchus infected by RMIV was found

to be much greater than previously reported (Matta and Lowe, 1970). It

was possible to detect light infections in some tissues by electron

microscopy which could not be detected by light microscopic studies. It

was obvious from the present study that both techniques are required to

17









Table 1.--Incidence of RMIV in A. taeniorhynchus larvae.


Collection site Datea No. Examined No. Infectedb


North Key July 12 4,439 5

North Key July 17 1,338 2

North Key Aug. 8 3,551 1

Atsena Otie Key Aug. 8 3,500 4


Totals 12,828 12


a The collection made on June 17, was not included in the table
because the number of larvae examined was not known.
b
Only larvae which exhibited iridescence were considered to be
infected.








adequately describe the pathology of the iridescent viruses. The following

tissues were found to be involved during infection.

Fat Body

The fat body tissue was the most extensively infected. Normal fat

body tissue was extensively vacuolated and contained numerous fat globules

(Fig. 1). In contrast, fat body which was infected was almost devoid of

any type of normal cytoplasmic structure, and contained large inclusions

which usually completely filled the cytoplasm of the infected cells.

These inclusions stained intensively with hematoxylin (Fig. 2) and the

Feulgen technique, indicating the presence of high concentrations of both

protein and DNA. Electron micrographs showed these inclusions to be large

numbers of closely packed virus particles. In many fat body cells the

virus particles were arranged in striking paracrystalline arrays (Fig. 3).

Arrays of this type are responsible for the iridescent colors produced by

MIV and the other iridescent viruses (Williams and Smith, 1957). The

virus particles in many fat body cells were not arranged in arrays (Fig.

4); the reason for this is not known. The term fat body is actually

misleading, since this tissue is an important storage depot for protein

and glycogen as well as for fat. These reserves are especially needed

during moulting and metamorphosis (Wigglesworth, 1953), and large amounts

of these nutrients are required for the moult to the pupal stage and for

the formation of imaginal structures prior to adult emergence. From

1 group of 700 fourth-instar larvae which were patently infected with

RMIV, only 69 (about 10 per cent) were capable of pupation, and only 1

developed to the adult stage. It was probably the destruction or disruption

of metabolic processes of the fat body by RMIV which prevented most of

the infected larvae from developing to the adult stage.



































Figure 1. Section of healthy A. taeniorhynchus larva; note vacuolated fat
body (FB) and tracheal epithelium (TE). Heidenhain's hematoxylin
and eosin. 812X.







































Figure 2. Section of infected A. taeniorhynchus larva showing densely
staining cytoplasmic inclusions in fat body (FB) and tracheal
epithelium (TE). Heidenhain's hematoxylin and eosin. 812X.










































te.

0













CI.




































Figure 3. Fat body cells with virus packed in paracrystalline arrays.
4,800X.





















; ...
s .^ .,.
'we* t-~


* .



































Figure 4. Infected fat body (FB) and tracheal epithelium (TE). Virus
particles not packed in arrays. 3,000X.












Epidermis

The extent of infection of the epidermis was variable within an

individual. Many cells contained large numbers of virus particles, while

others were only lightly infected (Fig. 5). Virus arrays in the epidermis,

as well as those found in the fat body, are responsible for the iridescence

exhibited by infected larvae when they are viewed against a dark background.

The tracheal epithelium, which is composed of modified epidermal tissue,

was usually greatly infected (Fig. 4). The significance that this infection

may have on oxygen uptake by infected larvae is unknown.

Other tissues of epidermal origin infected by RMIV were the imaginal

discs (Fig. 6). These discs appear in fourth-instar larvae as invaginations

of the epidermis in the thorax and the head, and are the developing anlage

of the imaginal wings, legs and antennae. Many cells of the imaginal

discs were greatly infected and it was doubtful whether appendages develop-

ing from these discs would have been functional even if the larvae had

been capable of developing to the adult stage. However, the single adult

which was successfully reared from a larva known to be patently infected

appeared to have completely functional appendages. There was no way to

evaluate the extent of infection in the imaginal discs from this specimen

during the larval stage.

Alimentary Tract

The alimentary tract of larval mosquitoes is very heterogeneous in

cellular structure. It originates anteriorly with the preoral cavity

which opens through the mouth into the pharynx, which then leads into the

esophagus. Each of these structures is lined with a thin layer of cuticle.

The esophagus, also covered with cuticle, is invaginated, and at the




































Figure 5. RMIV in epidermal cell. 24,000X.







































Figure 6. Infected imaginal disc. 6,400X.





















;,i~~* *fFZ:nl
* : *..
*'.I:


V
0
4.sr.

0.l~d









anterior end of the invagination a small imaginal ring separates the

esophagus from the midgut. From the anterior end, the midgut consists

of the cardia, the gastric caecae, and the stomach. The cells of the

midgut are characterized by large polytene nuclei and the presence of a

border of microvilli. Small embryonic cells are interspaced between

the cells of the stomach. A chitinous peritrophic membrane extends from

the cardia through the stomach. This membrane is continually replaced

and encloses the contents of the midgut. The hindgut begins posterior

to the stomach, and from the anterior end is composed of the malpighian

tubules, a posterior imaginal ring, the pyloric chamber, the small

intestine, the rectum, and the anal canal. The hindgut, with the

exception of the malpighian tubules and rectum, is lined with cuticle

similar to the foregut. The malpighian tubules and rectum are lined

with microvilli and are composed of cells with large polytene nuclei

similar to the midgut.

After reviewing the structure of the alimentary tract, the midgut

appeared to be the most likely site for the virus to enter the hemocoel.

Three-day-old larvae were fed high concentrations of virus (100 infected

larvae triturated in 20 ml of H20) and fixed for electron microscopy at

intervals of 30, 120, and 240 minutes. Examination of the midguts from

these larvae failed to reveal the presence of virus. However, the

possibility was not ruled out that a small number of virus particles

might enter by this route.

An extensive examination of the total alimentary tract by both light

and electron microscopy was initiated to attempt to identify tissues

which were susceptible to RMIV. Examination of the midgut, malpighian

tubules, and rectum failed to establish the presence of infection in










these tissues. The existence of heavy infections in imaginal disc tissues

suggested the possibility that the embryonic tissue of the gut might be

susceptible to infection by RMIV. Since the anterior imaginal ring was

the easiest embryonic tissue to find in the alimentary tract, it was

examined with the electron microscope. The anterior imaginal ring itself

was not infected; but, surprisingly, the esophageal tissue adjacent to it

did contain virus (Fig. 7). Normally the cuticular lining of the

esophagus would be expected to act as a mechanical barrier to infection

of this tissue by way of the alimentary canal, but any abrasion of the

cuticle could provide a possible mode of entry for the virus. This

possibility would be even more likely during the moulting process when

the new cuticle would be soft.

Another possible route of entry for the virus, which was not ruled

out, is penetration of the midgut by naked viral DNA if it is uncoated

by either high pH or proteolytic enzymes. The pH of the midgut of A.

taeniorhynchus is not known, but Clements (1963) listed values of 9.0 -

9.4 for Aedes albopiotus (Skuse). These pH values would probably not be

high enough to uncoat the DNA since RMIV preparations which were treated

at pH 10.5 for immunodiffusion studies did not appear to be completely

disrupted when examined with the electron microscope.

The theory of penetration by the naked DNA has several attractive

features. First, the naked DNA would be of a size which could readily

pass through the peritrophic membrane. Second, specific cellular receptor

sites on the cells of the midgut would not be required for penetration by

the viral DNA. Third, the low percentage of infection could be accounted

for by the decreased efficiency of infection by naked nucleic acids as


































Site of viral replication in a cell of the esophageal invagin-
ation; note presence of cuticle (CU) and microvilli (MV).
6,900X.


Figure 7.












compared to complete virions. These explanations are purely speculative

at the present time.

Hemocytes

Phagocytosis of pathogens by hemocytes is one of the primary

defensive responses of insects, and susceptibility of hemocytes

to infection by RMIV may serve to enhance the spread of virus throughout

the hemocoel. Only a few hemocytes were examined by electron microscopy.

One of these (Fig. 8) contained a small number of virus particles which

appeared to be enclosed in some type of vacuoles. These may have been

phagocytic vacuoles but no limiting membranes were evident. Some of the

hemocytes observed in tissue sections stained with hematoxylin or by the

Feulgen technique appeared to be heavily infected. It was impossible to

determine whether these cells were actually infected because hemocytes

phagocytize cellular debris which might also stain by these techniques.

It is doubtful, however, that phagocytized debris would stain as intensely

as viral inclusions.

Nerve

The visceral nerves were lightly infected by RMIV (Fig. 9), but no

tissue destruction was obvious. It was surprising that even though

visceral nerves were found to be infected, virus particles were never

observed in either abdominal or thoracic ganglia of the ventral nerve

cord. The ganglia were surrounded by a sheath of small cells (probably

neurosecretory cells) which were not infected, and these cells may have

served as a mechanical barrier to infection of the ganglia. The absence

of any extensive pathology in nerve tissue and the presence of extremely

light infections of muscle tissue may account for the ability of most

infected larvae to swim normally until shortly before death.



































Figure 8. Hemocyte with virus particles enclosed in vacuolar structures.
6,900X.








































Figure 9. Infected nerve. Axons (AX) appear normal. 9,200X.












Gonads

The testes of larval A. taeniorhynchus were never found to be infected

even though the layer of fat body tissue which surrounded them was invariably

heavily infected (Fig. 10). In females, a small number of virus particles

was observed within the nucleated sheath which surrounded the larval

ovaries (Fig. 11). The presence of virus in the larval ovaries was surprising

since Linley and Nielsen (1968a reported that progeny of mosquitoes developing

from patently infected larvae were not infected. The susceptibility of

larval gonads of A. taeniorhynchus to RMIV differs from results found with

A. atimulwns to its iridescent virus (Anderson, 1970). He reported that a

few cells of the testes were infected and that the ovaries were heavily

infected.

The electron microscopic examination of ovaries from adult A.

taeniorhynchus (treated as 3 day old larvae), which had produced infected

progeny, revealed the presence of numerous virus particles. Some of these

particles were in closely packed groups suggesting that ovarian tissue was

an active site of viral replication. Most of the virus was located in

cells of the follicular epithelium, and virus particles were also observed

in the nurse cells of these follicles. The presence of virus within the

follicles supports the contention of Woodard and Chapman (1968) and Linley

and Nielsen (1968a) that the virus is actually transmitted by infected

females to their progeny within the egg.

Transovarial Transmission

Adult female A. taeniorhynchus do not oviposit readily in small

containers; and consequently, eggs were obtained from only 24 mosquitoes.

Of the eggs from these 24 females, only 17 groups hatched. A total of

2,677 larvae from control mosquitoes failed to show any infection. The




































Figure 10. Larval testis (LT) of A. taeniorhynchus surrounded by infected
fat body (FB). 4,800X.



























*IO AI'
*.*. *. r S'
~bi *.*1te;.* *...0

*. 2 iii, *
ar~~~~ 1*~ -4,-: Vt ~.
*~*-



































Figure 11. Virus in nucleated sheath (NS) of larval ovary. 18,000X.





























































































t*Li:'~ 9 '$

.4


- ..-.


. p~









results of this experiment are given in Table 2. The results support the

assertion of Linley and Nielsen (1968a) that either all or none of the

progeny from a given mosquito are patently infected. The results also

suggest that if a mosquito is infected early, a patent infection always

results.

Irmunodiffusion

Purified, undegraded viruses did not diffuse readily in agar gels

and this resulted in a very dense precipitate at the edge of the antigen

well in immunodiffusion studies. The techniques used for degradation of

the viruses produced varying results. Virus treated with 0.05 N NaOH

usually formed only a single band in immunodiffusion tests, but occasionally

a very faint second precipitin band was apparent. Cunningham and Tinsley

(1968) were able to demonstrate 4 precipitin bands with TIV which had

been treated with 0.05 N NaOH, and it appears that some of the antigens of

MIV are destroyed by this treatment.

Virus preparations which were dialyzed against 0.2 M 2-aminoethanol

(pH 10.5) or 0.2 M Tris hydroxymethyll) aminomethane (pH 10.5) resulted

in the formation of 3 precipitin bands in agar gels using the 7 well

pattern. One of these was a very dense band which formed close to the

antigen well. The other 2 bands were more faint and formed approximately

midway between the antigen and antiserum wells. The faint bands were not

visible when the antigen preparation was diluted 1:8 with 0.85 per cent

NaCl. The bands formed by 0.2 M Tris treated virus were not as dense as

those produced by virus treated with 0.2 M 2-aminoethanol (pH 10.5).

Four bands were produced by virus which had been treated with 0.2 M 2-

aminoethanol (pH 11). The comparison of RMIV to TMIV treated in this

manner is presented in Figure 12. All precipitin bands formed reactions









Table 2.--Transovarial transmission of RMIV


Female No. No. of Progeny No. Patently Infected


1 1 1

2 4 0

3 156 156

4 20 0

5 16 0

6 18 0

7 5 0

8 66 66

9 23 0

10 175 0

11 4 0

12 13 0

13 24 0

14 167 0

15 167 0

16 168 0

17 197 0






49

of identity, although it is impossible to see the junction of the outer-

most bands in the photograph. Immunodiffusion plates utilizing the

concentrated crude homogenate of infected larvae resulted in the same

pattern as virus treated with 0.2 M 2-aminoethanol (pH 11).

There was a maximum of 2 bands produced in the 4 well square pattern

by both the concentrated crude homogenate and the 0.2 M 2-aminoethanol (pH 11)

treated virus. When using the square pattern, RMIV and TMIV were compared

simultaneously by placing each virus diagonally opposite its homologous

antiserum. Precipitin bands formed by common or related antigens were

continuous between the antigen and antibody wells. No unique antigens

were detected in any of the tests. Antigens unique to either virus would

have been recognized by the presence of precipitin bands running in a

direction parallel to the antigen-antibody axis of the other system. A

comparison of RMIV and TMIV using this pattern is shown in Figure 13.

Apparently not all of the proteins in MIV were evident in immuno-

diffusion studies. Wrigley (1969) reported finding at least 10 different

proteins by electrophoresis of disrupted SIV in polyacrylamide gels,

but Cunningham and Tinsley (1968) were able to demonstrate only 3 precipitin

bands with alkaline degraded SIV in immunodiffusion studies. On the basis

of these results, it is apparent that the iridescent viruses must contain

proteins which are not detectable by standard immunodiffusion techniques.

The inability to detect the proteins by these techniques could be due to

either the proteins being present in low concentrations or possibly to an

inherent low level of antigenicity. It is also possible that the dense

precipitin bands are actually composed of several bands produced by antigens

having very similar diffusion coefficients. As many as 7 bands were seen

in some studies with RMIV and TMIV after prolonged incubation, but some
























Figure 12.

























Figure 13.


Serological comparison of RMIV and TMIV using concentric
pattern. (1) buffer control, (2) host protein control,
(3) TMIV, (4) RMIV, (AS) anti-RMIV serum.























Serological comparison of RMIV and TMIV using the 4 well
square pattern. (1) anti-RMIV serum, (2) TMIV treated with
NaOH, (3) anti-TMIV serum, (4) RMIV treated with NaOH.







OPPFFSI









of these were probably due to secondary precipitation of the type described

by Crowle (1961). Comparisons of RMIV and TMIV by a more sensitive

technique, such as immunoelectrophoresis, might solve this problem, but

such a comparison was not performed.

Physical Comparisons of IRIV and TMIV

RMIV and TMIV sedimented at different rates when centrifuged in

sucrose density gradients (Fig. 14). A mixture of the 2 viruses in a

single tube formed 2 distinct bands during sedimentation in sucrose

gradients. The top band of the mixture corresponded to the position of

the TMIV band spun individually in another gradient tube, while the bottom

band corresponded to the position of the RMIV band. When these bands

were collected separately, diluted with phosphate buffer, and pelleted,

the iridescent pellet formed by virus from the upper band was a typical

TMIV pellet, while that from the lower band was a typical RMIV pellet.

Since both RMIV and TMIV particles appeared to be icosahedral on the

basis of electron micrographs, the greater rate of sedimentation of RMIV

was attributed to greater density and/or greater size. Subsequently, the

viruses were found to band at different positions in cesium chloride

gradients with the RMIV banding in a slightly lower position. This

indicated that RMIV is slightly more dense than TMIV. Measurements from

electron micrographs of tissue sections demonstrated that RMIV is also

larger in diameter than TMIV (Table 3). The averages of 20 side-to-side

measurements were 195 mp for RMIV and 160 my for TMIV. These r-sults posed

the interesting but still unanswered question of how the substituents of

these viruses, which are identical in major antigenic components, are

assembled to produce virions which are so different in size. Ultra-

structural studies utilizing the technique of Wrigley (1969) were attempted

but were unsuccessful.



































Figure 14. Comparison of RMIV and TMIV in sucrose density gradients.
(R) RMIV, (T) TMIV.




54





5






55

Table 3.--Side-to-side measurements of PMIV and TMIV from electron
micrographs of tissue sections.




Number RMIV TMIV


201

194

191

197

201

201

191

197

191

194

194

188

197

184

194

191

197

194

201

197


164

168

174

161

164

161

158

164

158

164

155

171

161

158

155

151

151

148

151

155


Average = 1954.5 Average = 1606.8









During electron microscopic examination of tissues infected with

RMIV and TMIV, tubular structures which were associated with the viruses

were conmnonly observed (Fig. 15). These tubes appeared to be fairly uniform

in diameter and slightly smaller than the respective virus with which they

were associated. The fact that the tubes associated with RMIV were larger

than those associated with TMIV suggested that they were of viral origin.

It was also of interest that many of the virus particles in the vicinity

of the tubes appeared to be incomplete and without cores, which suggested

that the tubes may have been the result of a faulty assembly mechanism.
*
Caspar and Klug (1962) postulated that the most probable mistake in the

assembly of an icosahedral virus would lead to tubular forms. Similar

tubular structures have been seen in association with other icosahedral

viruses. Hitchborn and Hills (1968) presented morphological, serological,

and chemical evidence that both the virus particles of necrotic turnip

yellow mosaic virus and the tubes associated with this virus were composed

of the same protein subunits. Further evidence for the viral origin of

these tubular structures was the finding by Bancroft et al. (1967) that

the substituents of cowpea chlorotic mottle virus will recombine under

certain conditions in vitro to form tubes. Similar tubular structures

have been reported in association with polyoma virus (Howatson and

Almeida, 1960), papilloma viruses (Williams et al., 1960; Finch and Klug,

1965), human wart virus (Noyes, 1964), rabbit kidney vacuolating virus

(Chambers et at., 1966), and a frog virus (Darlington et al., 1966).

RMIV and TMIV each produce a distinct iridescence in infected larvae.

Also, the viruses were readily distinguished by the iridescence of their

purified pellets (Figs. 16 and 17). When pelleted in distilled water,

purified RMIV produced a pink pellet and TMIV produced a light green pellet.



































Figure 15. Tubular structures in imaginal disc tissue infected with RMIV.
9,600X.







58















see .1C P",

Alp
40






59


Figure 16.


Purified pellet of RMIV (Sedimented from 0.1 M NaCI in 0.01 M
borate buffer).






60


Figure 17.


Purified pellet of TMIV (Sedimented from 0.1 M NaCI in 0.01 M
borate buffer).









As the pellets were dehydrated by ethanol, the color of iridescence

produced by each virus shifted toward the shorter wavelengths. The same

effect resulted from pelleting the viruses in increasing concentrations

of NaC1 up to 0.08 M, but at concentrations above this point very little

change in color was noticeable. In 0.08 M NaCl, the iridescence produced

by RMIV was almost identical to that of TMIV in distilled water. These

results suggested that the forces acting in the paracrystalline arrays

might be due to charges on the virus. The effect that pH may have on

the iridescence was not studied. The observation that RMIV and TMIV can

be distinguished on the basis of the iridescence of purified pellets, and

the discovery that mixtures of the 2 viruses can be separated by sucrose

density gradient centrifugation, should be valuable in studies of mixed

infections.














SUMMARY AND CONCLUSIONS


RMIV was found to be endemic at low levels in natural populations of

A. taeniorhynchus from the west coast of Florida. This virus was highly

virulent once the infection process was initiated. Only a very small

percentage of patently infected larvae were capable of developing to the

adult stage. The purpose of this study was to describe the pathology and

biology of RMIV in A. taeniorhynchus and to determine the relationship

between RMIV and TMIV.

The fat body, epidermis, tracheal epithelium, and imaginal discs

were the primary sites of viral replication in A. taeniorhynchus larvae.

The cytoplasm of nearly all of the cells of the fat body and tracheal

epithelium was completely destroyed. 'It was concluded that the destruction

of the fat body was responsible for the mortality in larvae and pupae.

The extent of pathology in the epidermis and imaginal discs varies from

cell to cell. Other tissues which were infected to a lesser extent were

the esophagus, hemocytes, nerves, muscle, and larval ovaries. There was

no obvious pathology in these tissues.

The route of entry of RMIV into the hemocoel was not definitely

established. However, the presence of virus in the esophagus suggested

that this tissue could be involved if the cuticular lining is abraded.

One problem which was not explained was the failure of Linley and

Nielsen (1968a) to demonstrate patent infections in progeny from females

which developed from patently infected larvae. This problem was made

62










more perplexing by the demonstration during the present study of virus

in the developing ovaries of patently infected larvae. Unfortunately,

the author was unable to rear patently infected larvae to the adult stage

and examine their progeny.

The transovarial transmission of RMIV by females which were exposed

to virus as third- or fourth- instar larvae was substantiated. The

supposition of Linley and Nielsen (1968a) that the virus is transmitted

to either all or none of the progeny of a given female was also sub-

stantiated. When the ovaries of mosquitoes which had produced infected

progeny were examined, virus was observed in both the follicular epithelium

and nurse cells.

The relationship between RMIV and TMIV was studied by comparing the

serological and physical properties of the viruses. Four antigens were

detected in gel diffusion studies with degraded virus preparations all of

which were common to both viruses. No unique antigens were detected for

either virus. Tubular structures were found associated with both RMIV

and TMIV in electron micrographs of tissue sections. These structures

were slightly smaller in diameter than the respective virus with which

they were associated. If these tubes are composed of viral protein

subunits, as are similar tubes associated with turnip yellow mosaic virus,

the fact that the tubes associated with RMIV are different in size from

those associated with TMIV suggests that the proteins of the virus are

different. The difference in iridescence produced by the 2 viruses is

probably also a manifestation of a difference in the structural proteins.

A more sensitive technique will probably be required to demonstrate an

actual difference between the proteins of the 2 viruses.






64

The observation was made that RMIV and TMIV sedimented at different

rates in sucrose density gradients. Subsequently, RMIV was found to be

slightly more dense than TMIV by equilibrium ultracentrifugation in

cesium chloride. RMIV was also found to be considerably larger than

TMIV on the basis of measurements from electron micrographs.




































APPENDICES















Appendix 1. Staining Schedules for Light Microscopy



Heidenhain's Hematoxylin

1. Remove paraffin with xylene.

2. Hydrate to distilled water.

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

4. Stain overnight in 0.25 per cent Heidenhain's hematoxylin.

5. Rinse in running water for 5 minutes.

6. Differentiate in 2.5 per cent iron alum.

7. When differentiated, 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 30 minutes in 0.5 per cent erythrosin B.

11. Dehydrate rapidly to absolute ethanol, clear in xylene and mount.

















1, Humason, 1967.










Feulgen Reaction

1. Remove paraffin with xylene.

2. Hydrate to distilled water (leave in 95 per cent ethanol overnight

to remove lipids).

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

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

5. Rinse at room temperature in N HC1 for 1 minute.

6. Rinse in distilled water.

7. Stain in Schiff's reagent for 2 hours in dark.

8. Drain and transfer quickly into bleaching solution, 3 changes for

2 minutes each.

9. Wash in running water for 15 minutes.

10. Rinse in distilled water.

11. Counterstain in 0.05 per cent fast green for 1 minute.

12. Dehydrate rapidly to absolute ethanol, clear in xylene, and mount.














Appendix 2. Fixation and Embedding Schedule for Electron Microscopy



1. 3 per cent glutaraldehyde 3 hours (room temperature).

2. Rinse in 0.1 M phosphate buffer overnight at 4C.

3. 1 per cent osmium tetroxide 2 hours.

4. 0.1 M phosphate buffer 15 minutes.

5. Dehydrate in ascending alcohol series.

6. Propylene oxide, 2 changes 1 hour each.

7. Epon and propylene oxide (equal parts) overnight in refrigerator.

8. Pure epon, 2 changes 4 hours and overnight.

9. Embed in epon.














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* Original not seen.














BIOGRAPHICAL SKETCH


Donald William Hall was born December 11, 1942 at Muncie, Indiana.

He was graduated from Muncie Central High School in June, 1960. He

received the degree of Bachelor of Science in Agriculture in June, 1964,

from Purdue University with a major in entomology. In February, 1965,

he enrolled in graduate school at Purdue University, where he received

the degree of Master of Science with a major in entomology in June, 1967.

From September, 1967, until the present time he has worked toward the

degree of Doctor of Philosophy at the University of Florida.

Donald William Hall is married to the former Diane Jeanne Armour and

is the father of one daughter, Lisa Anne.















This dissertation was prepared under the direction of the chairman

of the candidate's supervisory committee and has been approved by all

members of that committee. It was submitted to the Dean of the College

of Agriculture and to the Graduate Council, and was approved as partial

fulfillment of the requirements for the degree of Doctor of Philosophy.

August, 1970.






a, College of Agriculture

i" l


Dean, Graduate School


Supervisory Committee:


Chairman




Co-Chairman



S.i


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q1/ ) A


Internet Distribution Consent Agreement

In reference to the following dissertation:

Author: Donald William Hall


Title: The Pathobiology of a Mosquito Iridescent Virus in Aedes taeniorhynchus
(Wiedemann)


Publication Date:


1970


I, Donald Willliam Hall, as copyright holder for the aforementioned dissertation, hereby grant
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Florida and its agents. I authorize the University of Florida to digitize and distribute the
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This grant of permissions prohibits use of the digitized versions for commercial use or profit.




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April 1, 2008
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1


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