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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Methods and materials
 Results and discussion
 Summary
 Bibliography
 Biographical sketch














Group Title: regular mosquito iridescent virus (RMIV) in the black saltmarsh mosquito, Aedes taeniorhynchus (Wiedemann): production, purification, transovarial transmission, site of entry, development of infections, and polypeptide composition /
Title: The regular mosquito iridescent virus (RMIV) in the black saltmarsh mosquito, Aedes taeniorhynchus (Wiedemann): production, purification, transovarial transmission, site of entry, development of infections, and polypeptide composition /
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 Material Information
Title: The regular mosquito iridescent virus (RMIV) in the black saltmarsh mosquito, Aedes taeniorhynchus (Wiedemann): production, purification, transovarial transmission, site of entry, development of infections, and polypeptide composition /
Physical Description: 126 leaves : ill. ; 28 cm.
Language: English
Creator: Hembree, Stephen Carson, 1938-
Publication Date: 1974
Copyright Date: 1974
 Subjects
Subject: Viruses   ( lcsh )
Mosquitoes   ( 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, 1974.
Bibliography: Includes bibliographical references (leaves 119-124).
Statement of Responsibility: by Stephen Carson Hembree.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098171
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000430669
oclc - 38007210
notis - ACJ0026

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Table of Contents
    Title Page
        Page i
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
        Page vi
        Page vii
    List of Tables
        Page viii
    List of Figures
        Page ix
        Page x
    Abstract
        Page xi
        Page xii
        Page xiii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
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        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Methods and materials
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
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        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
    Results and discussion
        Page 62
        Page 63
        Page 64
        Page 65
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        Page 68
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        Page 113
        Page 114
    Summary
        Page 115
        Page 116
        Page 117
        Page 118
    Bibliography
        Page 119
        Page 120
        Page 121
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        Page 123
        Page 124
    Biographical sketch
        Page 125
        Page 126
        Page 127
        Page 128
Full Text











THE REGULAR MOSQUITO IRIDESCENT VIRUS (RMIV) IN THE
BLACK SALTMARSH MOSQUITO, AEDES TAENIORHYNCHUS
(WIEDEMANN): PRODUCTION, PURIFICATION,
TRANSOVARIAL TRANSMISSION, SITE OF ENTRY,
DEVELOPMENT OF INFECTIONS, AND
POLYPEPTIDE COMPOSITION









By

STEPHEN CARSON HEMBREE


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
1974


































Dedicated to Jane, my wife















ACKNOWLEDGMElT'S


I wish to express gratitude to my suIrvisory com-

mittee: Dr. F. S. Blanton, Dr. R. E. Lowc-, Dr. S. G. Zam,

Dr. J. L. Nation, and Dr. F. W. Zettler.

Appreciation is also expressed to Dr. D. L. Wiedhaas,

Director, and to the staff of the Insects Affecting Man

and Animals Research Laboratory, U. S. Department of Agri-

culture, for their support of this proje-c. I wish

especially to thank Mr. D. W. Anthony, for assistance with

electron microscopy, and Mrs. A. L. Camoron, Mr. H. R.

Ford, and Mr. E. Green for assistance in many ways.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS . . .

LIST OF TABLES . . . .


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

ABSTRACT . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . .


METHODS AND MATERIALS.


viii
viii


. . . . . . . . 21


Production of Virus


Routine Virus Production procedure ... . 21
Determination of Optimum Quantity
of RMIV Inoculum . . . .. . .. 25
Determination of the Optimum Larval
Age for Exposure to RMIV . . . .. 26
Purification of RMTV . . . . . .. 27
Quantification of RMIV Production. ... . 31
Attenuation of Infectivity of RMIV
During Purification. . . . . ... 32

Transmission of RMIV. . . . . . . ... 33


Per Cent of Larvae Infected with
RMIV by Transovarial Transmission.
Transovarial Transmission Through
Isolated Females . . . ..
Sex Specificity of Transovarial
Transmission of RMTV . . .
Transmission of RMIV by
Integument Puncture . . .


. . 33

. . 34

. . 36

. . 37









TABLE OF CONTENTS (Continued)


Page

Production of Antiserum to RMIV and
to A. taeniorhynchus Antigens. . . . .. 38
Purification of Gamma Globulin . . . ... 40
Conjugation of Anti-RMIV Antibody with
Fluoresceine Isothiocyanate (FITC) . . .. 41
Pilot Project on Autoradiography of
3H-Mothyl Thymidine Uptake and Dis-
tribution in Larval A. taeniorhvnchus. ... . 46
Production of 3H-Methyl Thymidine
Labeled RMIV . . . . . . . . 51
Site of Entry of RMIV into Host Tissue . . 52

Light Microscope Studies. . . . .. 52

Autoradiography. . . . . ... 52
Fluorescent antibody . . . ... 53

Electron Microscope Studies . . . .. 53

Development of RMIV Infections . . . ... 55

Autoradiography ........ . . . .55
Fluorescent Antibody. . . . . ... 56

Overt infections . . . . .. 56
Covert infections. . . . . ... 57

Number and Molecular Weights of RMIV
Structural Polypeptides. . . . .... . 58
Isolation of RMIV Structural Polypeptides
by Hydroxylapatite Chromatography. . . .. 60

RESULTS AND DISCUSSION . . . . . . ... .62

Production of RMIV . . . . . . ... 62

Determination of Optimum Quantity
of RMIV Inoculum. . . . . . .. 62









TABLE OF CONTENTS (Continued)


Determination of Optimum Larval
Age for Exposure to RMIV. . . . .. 65
Quantification of RMIV Production . . . 65
Attenuation of Infectivity of
RMIV During Purification. . . . .. 68

Transmission of RMIV . . . . . . .. 70

per Cent of Larvae Infected with
RMIV by Transovarial Transmission . . 70
Transmission of RMIV Through
Isolated Females. . . . . . .. 72
Sex Specificity of Transovarial
Transmission. . . . . . . .. 73
Total of Overt and Covert
Transmission. . . . . . . . 75
Transmission of RMIV by Integument
Puncture. . . . . . . . . 78

Pilot Project on Autoradiography of
3H-Methyl Thymidine Uptake and
Distribution in Larval A. taeniorhvnchus . 79
Site of Entry of RMIV Into Host Tissues. ... . 82

Light Microscope Studies. . . . . .. 82

Autoradiography. . . . . . .. 82
Fluorescent antibody . . . . . 86

Electron Microscope Studies . . . .. 86

Development of RMIV Infections . . . . .. 97

Autoradiography . . . . . . .. 97
Fluorescent Antibody. . . . . . ... 100

Overt infections . . . . . .. 100
Covert infections. . . . . . 107









TABLE OF CONTENTS (Continued)


page

Number and Molecular Weights of
RMIV Structural Polypeptides. . . . .. 110
Separation of RMIV Structural
Polypeptides by Hydroxylapatite
Chromatography. . . . . . . . .. 114

SUMMARY . . . . . . . . . . . .. 115

BIBLIOGRAPHY . . . . . . . . .. .. . 119

BIOGRAPHICAL SKETCH. . . . . . . . .. 125















LIST OF TABLES


Table Page

1 Net Virus Production Resulting from
24 Hr Exposure of Day Old A. taeniorhvnchus
Larvae to RMIV. . . . . . . .. 63

2 RMIV Production Resulting from 24 Hr
Exposure of A. taeniorhynchus Larvae
of Different Ages to 50 LEQ of
Inoculum. . . . . . . . . . 66

3 Attenuation of Infectivity Resulting from
purification of RMIV Through the Stage
Indicated . . . . . . . . . 969

4 Transovarial Transmission of RMIV by
Reciprocal Mating of Exposed and Unex-
posed Male and Female A. taeniorhynchus . 74

5 Total of Overt and Covert Transmission
of RMIV to Larvae Exposed at Different
Ages .................. . 77

6 Proteins Used as Markers for the Estimation
of the Molecular Weights of RMIV Struc-
tural Polypeptides. ........... 111

7 Relative Migration Fractions and Molecular
Weights of RMIV Structural Polypeptides 112


viii















LIST OF FIGURES


Figure .iaLe

1 per Cent Transmission of RMIV in
A. taeniorhvnchus Larvae Exposed to
Increasing Amounts of Inoculum. . . .. 64

2 per Cent of RMIV Infection Among Progeny
of Mosquitoes Exposed to RMIV as Larvae
of Different Ages . . . . . ... .71

3 Malpighian Tubule Cell with Labeled
Nucleus . . . . . . . . ... .80

4 Anterior Midgut Region of Larva Showing
Residual Deposits of Tritiated Thymidine
Peripheral to Caudal Extremity of Foregut
Invagination into Midgut. . . . . .. 83

5 RMIV Infected Fatbody Cell in A.
taeniorhynchus Larva with Graination
Over Cytoplasmic Deposits of Tritiated
Thymidine Labeled Viral DNA . . . .. 85

6 Midgut Epithelium Cell Containing
Scattered RMIV particles. . . . . ... 88

7 Midgut Epithelium Cell with Disarranged
Microvilli and Containing Scattered RMIV
particles . . . . . . . ... .89

8 RMIV Particles pressed Against Peritrophic
Membrane in Midgut of A. taeniorhynchus . 90

9 RMIV particles pressed Against Peritrophic
Membrane in Midgut of A. taeniorhvnchus . 91









LIST OF FIGURES (Continued)


Figure


Page


10 RMIV Particle Between peritrophic
Membrane and Midgut Epithelium. . . .

11 Virus Like Particle Apparently Within
a Microvillus . . . . . . .

12 Small Pocket of RMIV in Non-Epithelial
Cell in Midgut Wall of A. taeniorhynchus
Larva . . . . . . . . .

13 Methyl Green Staining of RMIV Infected
Cells . . . . . . . . .

14 Fluorescent Antibody Staining of Epidermal
Cells, Imaginal Bud Tissues and Fatbody
Tissue Infected with RMIV . . . .

15 Fluorescent Antibody Staining of RMIV
Infected Tracheal Epithelium Cell . .

16 Fluorescent Antibody Staining of Fatbody
Surrounding Testis. . . . . . .


. 92



. 93




. 95



. 99




. 102



. 103



. 105









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 REGULAR MOSQUITO IRIDESCENT VIRUS (RMIV) IN THE
BLACK SALTMARSH MOSQUITO, AEDES TAENIORHYNCHUS
(WIEDEMANN): PRODUCTION, PURIFICATION,
TRANSOVARIAL TRANSMISSION, SITE OF ENTRY,
DEVELOPMENT OF INFECTIONS, AND
POLYPEPTIDE COMPOSITION

By

Stephen Carson Hembree

March, 1974

Chairman: Franklin S. Blanton
Major Department: Entomology and Nematology

A system permitting the weekly production by 1 person

of gram quantities of the regular mosquito iridescent virus

(RMIV) in the black saltmarsh mosquito, Aedes taeniorhynchus

(Wiedemann), was described. The efficiency of the system re-

sulted from exposure of larvae at their most susceptible age

to an optimum quantity of RMIV inoculum and from the use of

a virus purification procedure designed to minimize waste.

Transmission rates of about 10 per cent were routinely

achieved. An average of 58 pg of virus was produced per in-

fected larva, constituting 9.35 per cent of the dry weight of

the average infected larva. Attenuation of infectivity oc-

curred throughout the virus purification procedure, no

major part of it being attributable to a single cause.









Transovarial transmission of the virus increased with

the age of larvae at exposure through day 5 of larval

life, but decreased when exposure was made just before

pupation. All of the larvae produced by 9 isolated,

transovarially transmitting females were infected. Trans-

mitting females produced fewer average progeny than non-

transmitting females. Males did not transmit virus to

previously unexposed females. Total transmission (i.e.,

transmission resulting in overt disease plus transmission

indicated only by infections among progeny) decreased with

the age of larvae at exposure and was greatest for larvae

in the second instar when exposed.

A probable site of entry of RMIV into host tissue was

found by electronmicroscopy in midgut epithelium at the

level of the foregut invagination. Infections were found

in fatbody, epidermis, imaginal buds and tracheal epithelium

by autoradiography of 3H-methyl thymidine treated larvae

84 hr after the initiation of exposure to the virus. In-

fections were found in fatbody by fluorescent antibody

staining 48 hr after initiation of exposure. Infection had

appeared in epidermis and in imaginal buds by 72 hr after

initiation of exposure, and in tracheal epithelium by 96

hr after initiation of exposure. Infections resulting in









transovarial transmission could not be detected by fluorescent

antibody staining.

Twenty-five polypeptides were found in RMIV by poly-

acrylamide gel disc electrophoresis of sodium dodecyl

sulfate--2-mercaptoethanol disrupted virus. The molecular

weights of these were estimated by polyacrylamide gel disc

electrophoresis and ranged from 23,500 to 331,000. Fourteen

per cent of the genome of the virus could code for its

structural polypeptides, assuming no redundancy and that

no 5S ribosomal RNA or transfer RNA were coded for.


xiii















INTRODUCTION


The relationships between viruses and arthropods are

of great interest and importance to man. Viruses are

etiologic agents of many diseases of man, of his domestic

animals, and of his crops. Arthropods are vectors for many

of these and serve also as reservoirs for some diseases

of both man and animals (Chamberlain, 1968) and of plants

(Maramorosch, 1968). This necessitates an especially inti-

mate relationship between the disease agents and their vectors.

Arthropods themselves are susceptible to other viral diseases

that are not known to infect either plants or other animals.

Most of these have been found affecting insects (Smith,

1967). These diseases are of particular interest in consi-

deration of current efforts to find non-chemical means

of reducing some arthropod populations. Although the

pathogenicity of the viral diseases of arthropods has long

been recognized, the realization that many arboviruses and

arthropod-transmitted plant viruses are pathogenic to

their vectors is relatively recent (Chamberlain, 1968;

Maramorosch, 1968). The relationships between viruses and








arthropods are now known to form a symbiotic spectrum

ranging from mutual benefit at one extreme to population

decimating lethal parasitism at the othe- (Maramorosch

and Jensen, 1963).

The viruses of insects can be divided into two groups

on the basis of whether the virions are enveloped in

protein crystals (occluded) or whether they are not (non-

occluded) (Smith, 1967, p. 8). Of the non-occluded insect

viruses, the iridescent viruses have attracted the atten-

tion of virologists for several reasons. They are the

largest symmetrical viruses known (Smith, 1967, p. 84);

they are produced in great relative quantity, composing up

to 25 per cent of the dry weight of their host (Williams

and Smith, 1957); they are probably the easiest of all

viruses to isolate and purify (Smith, 1967, p. 78); and

they are DNA containing viruses which replicate in the

cytoplasm of host cells (Wildy, 1971).

The International Committee on Nomenclature of Viruses

(ICNV) established at the International Congress for

Microbiology in 1966 placed the iridescent viruses in the

genus Iridovirus (Wildy, 1971). The members of this genus

were defined as large (130 180 nm), non-occluded,

icosahedral, DNA containing viruses which replicate in








the cytoplasm of the host cell. T;he Tiuula irridescent virus

(TIV), isolated from the leatherjacket, Tipula oaludosa

(Meigen)(Diptera), was designated as the type species.

Other members of the genus were Chilo iridescent virus

(CIV), from the rice stem borer, Chile suppressalis Walker

(Lepidoptera), and Sericesthis iridescent virus (SIV),

isolated from the pruinose scarab, Sericesthis oruinosa

(Dalman) (Coleoptera). The literature of these three viruses

recently has been well reviewed by Smith (1967) and Bellett

(1968). The mosquito iridescent virus (MIV), first described

from the black saltmarsh mosquito, Aedes taeniorhynchus

(Wiedemann), by Clark et al. (1965), was listed as a probable

member of the genus. The uncertainty shown at that time

was due to a lack of information of the physical and chemi-

cal characteristics of this virus which subsequently

became available (Matta, 1970; Matta and Lowe, 1970). At

the present time, no species names have been approved for

iridoviruses by the ICNV, and an informal nomenclature

that identifies the viruses by use of the name of the host

has gained acceptance in the current literature and has

been used in this dissertation.

The most conspicuous feature of the iridoviruses that

have been recognized by the ICNV is their characteristic









iridescence when viewed by reflected light. Visible color

is evident both in heavily infected host tissue and when

the purified virus is pelleted by ultracentrifugation.

This iridescence has been explained as Bragg reflection of

visible light from randomly oriented microcrystals of the

virus, the formation of which is made possible by the ex-

treme regularity of size and shape of the virions (Williams

and Smith, 1957; Smith and Williams, 1958; Klug et al.,

1959). This feature of the genus Iridovirus is shared with

a number of other insect viruses which could not be included

in the genus because too little was known of their charac-

teristics. The extent to which the phenomenon of iridescence

is correlated with the physical, biological and biochemical

characteristics which define the genus Iridovirus is un-

certain. It is conceivable that other viruses might be

found that iridesce but differ from the iridoviruses as

they have been defined. Several icosahedral cytoplasmic

deoxyriboviruses are known which do not form into the

paracrystalline arrays necessary for iridescence (Stoltz,

1971). Nevertheless, the iridescent viruses of insects

are generally considered to be closely related (Bellett,

1968; Bellett and Inman, 1967; Cunningham and Tinsley,

1968; Glitz et al., 1968; Wildy, 1971). Tinsley and Kelly





5


(1970) suggested an interim nomencl tcure sc:tem for this

group of viruses. Their published ist of 12 viruses

should now contain two addition!l- ors recc.,:tly reported

from a chaoborid, Corethrella brrAke...i., in Louisiana

(Chapman et al., 1971) and from a gras? grub, Costelytra

zealandica, in New Zealand (Kalmakoff eft al., 1972).

Iridescent viruses have been reported infecting several

species of Aedes mosquitoes and one of Psorophora from widely

separated localities:

Aedes taeniorhynchus (Wiedemann)
Florida (Clark et al., 1965; Hall and Lowe, 1971)
Louisiana (Chapman et al., 1966)

Aedes annulipes (Meigen)
Czechoslovakia (Weiser, 1965)

Aedes cantans (Meigen)
Great Britain (Tinsley et al., 1971)
Czechoslovakia (Weiser, 1965)

Aedes fulvus pallens (Ross)
Louisiana (Chapman et al., 1966)

Aedes vexans (Meigen)
Louisiana (Chapman et al., 1966)

Aedes dorsalis (Meigen)
Nevada (Chapman et al., 1966)

Aedes detritus (Haliday)
Great Britain (Service, 1968)
Tunisia (Vago et al., 1969)
France (Hasan et al., 1970)









Aedes stimulans (walker)
Connecticut (Anderson, 1970)

Psorophora ferox (Humb o]t)
Louisiana (ch3pma:i t al., 1966)

The relationship of these isolates is not completely

known. Cross infectivity stLdies were conducted among

the known hosts of mosquito iridescent viruses in Louisiana

(Woodard and Chapman, 1968), and in all tests the isolates

were infective for species other than their own host.

However, there was consistently less than 1 per cent in-

fectivity, compared to 15 per cent to 18 per cent infectivity

in the original hosts. These data suggest the viruses from

different species were not identical. Aedes sollicitans

(Walker) is frequently found in natural breeding sites with

A. taeniorhynchus, but has never been found infected with

RMIV in nature, even in the presence of infected A. taenior-

hynchus. However, it can be infected artificially in the

laboratory with about the same infectivity rate as the

natural host. The iridescent virus from A. stimulans is

apparently distinct from other MIV, since it is considerably

smaller (135 nm vs. 180 nm) and is lethal to the larvae in

the late third rather than the fourth stadium (Anderson,

1970). Isolates of MIV from A. detritus, collected in

Tunisia and Southern France, were compared morphologically








and serologically and were considered to be identical

(Hasan, et al., 1970). The iridescent virus from A. cantans

in Great Britain showed a relationship to that from

A. taeniorhynchus by the tube precipitin test but showed

no relationship to TIV, SIV, CIV, or to the iridescent

virus from Wiseana cervinata (Lepidoptera) in New Zealand

(Tinsley et al., 1971). In addition, the iridescent virus

from A. taeniorhynchus was unrelated to TIV or SIV when

tested by complement fixation (Cunningham and Tinsley,

1968).

Of the iridescent viruses reported from mosquitoes,

only the one from A. taeniorhynchus in Florida (Clark et al.,

1965) has been well characterized. Two strains of this

virus were reported from Louisiana. One exhibited a yellow

to green, and sometimes pink, iridescence and the other a

blue iridescence (Woodard and Chapman, 1968). The latter

has been collected only from Louisiana, and it has been

suggested that this isolate is not as prevalent as the

former (Hall and Lowe, 1971). The blue isolate was

designated the turquoise mosquito iridescent virus (TMIV),

and the orange isolate,the regular mosquito iridescent

virus (RMIV) (Matta and Lowe, 1970). The infectivity of

the 2 isolates was shown to be about the same (Woodard








and Chapman, 1968). Slightly mure DNA was present in

RMIV than in TMIV (Faust et al., 1968; Wagner et al.,

1973) and neither contained RNA (Faust et al., 1968).

RMIV and TMIV have been further compared physically,

chemically, and serologically (Lowe et al., 1970, Hall and

Lowe, 1972). Measurement of the virions of both types,

in tissue sections and after negative staining of purified

virus, indicated that RMIV was larger than TMIV. Also,

RMIV had a greater sedimentation rate in sucrose density

gradients and greater density in CsCl gradients. No

antigenic differences could be detected between the strains

at the level of sensitivity of agar gel diffusion tests.

Both strains infected the same host tissues and caused

similar cytopathology. Tubular structures of unknown origin,

slightly smaller in diameter than the virus they accom-

panied, were found in tissues infected with both strains

(Lowe et al., 1970; Hall and Anthony, 1971). A recent

study attributed several biophysical differences between

the strains to their relative sizes (Wagner et al., 1973).

Additionally, their electrophoretic mobilities were dif-

ferent, but their isoelectric points were similar. Dif-

ferences were shown in the percentages of protein, DNA,

and lipid, but structural differences other than size were

not detected.








Several physicochemical characteristics of RMIV have

been reported by Matta (1970). Spectronhotometric analysis

showed that the ratios of ultraviolet absorption to the

concentration of virus suspension were constant at a wave

1%
length of 700 nm, and an extinction coefficient (E%00)

of 10.8 was determined to permit easy quantification of

purified virus suspensions. The sedimentation coefficient

(S20'w) was 4458, and the density was calculated to be

1.354 g/cm3. The particle weight, calculated from the

diameter (180 nm) and density of the particle, was 4.129

x 10-15 g or 2.486 x 10 daltons. The DNA content of

15.97 Z 0.29%, determined by diphenylamine with a known

quantity of virus, was larger than the 11.7 per cent re-

ported previously by Faust et al. (1968) and accounted for

a DNA molecular weight of 397 x 106 daltons. Wagner et al.

(1973) reported a content of 16.9 t 0.1% DNA, and, using

this figure and the particle weight, they calculated the

molecular weight of the DNA to be 464 x 106 daltons. Both

of these figures for the molecular weight of DNA are more

than twice as large as those of any other known non-

iridescent virus (Wagner et al., 1973). The amino acid

composition was found to be very similar to that of SIV

and TIV. Wagner et al. (1973) also found lipid in RMIV,









but the possible structural role of this lipid, like that

found in TIV (Glitz et al., 1968), remains unresolved.

Some of these d&ita were used to differentiate RMIV from

other iridescent viruses (Lowe et al., 1970).

The structure of RMIV has been studied by high resolu-

tion electronmicroscopy as one representative of icosahedral

cytoplasmic deoxyriboviruses by Stoltz (1971). He reported

that the electron dense nucleoid was surrounded by a

trilaminar membrane, morphologically resembling a unit

membrane, that closely followed the contour of the nucleoid

and was thought to be a part of it.

Surrounding the nucleoid and the inner membrane was an

outer membrane, closely appressed to the latter in intact

virions. The contact line of the 2 membranes was electron

dense and twice as thick as a single membrane lamella.

In sectioned material there appeared to be a layer of

morphological subunits on the surface of the outer membrane,

but they could not be resolved by negative staining of

intact virions. The rigidity of these external structural

subunits was thought to be responsible for the icosahedral

shape of the virus. The outer membrane and morphological

subunits thereon were defined as the shell of the virus,

because they tended to maintain their characteristic angular








configuration in disrupted particles. The inner membrane

appeared flexible and could maintain the contour of either

the nucleoid or the shell. The inner membrane remained

attached to the nucleoid in disrupted particles more fre-

quently than to the shell. In a note appended to his paper,

Stoltz (1971) proclaimed that morphological subunits had

been detected by negative staining on the surface of

collapsed virions and that these were organized into

triangular shaped "trisymmetrons" (terminology of Wrigley,

1969, 1970) which were hexagonally arranged. Thus, the

structure of the shell of RMIV resembled that of SIV

(Wrigley, 1969) and TIV (Wrigley, 1970; Stoltz, 1971).

Clark et al.(1965) demonstrated that per os trans-

mission of MIV was possible, and they also reported that

the infectivity of the virus apparently was destroyed by

either drying or putrefaction. Woodard and Chapman (1968)

routinely transmitted the virus in the laboratory by

per os exposures for up to 68 serial passages with a mean

transmission rate of 16 per cent. Their studies also

indicated that various exposure methods influenced the

success of transmission of the virus. Larvae exposed to

RMIV for 24 hr during the first through the third, but not

the fourth, stadium demonstrated infection before pupation.

Optimal transmission appeared to occur when the larvae








were exposed during the second stadium. Transmission

increased with the duration of exposure up to 48 hr, but

additional exposure caused only a slight increase in per

cent transmission. When late first and early second

instar larvae were exposed for 24 hr to various numbers

of macerated, infected larvae, there was a sharp increase

in the transmission rate as the quantity of inoculum in-

creased but also a sharp increase in larval mortality.

However, transmission was quantitated on the number of

surviving larvae rather than the number of exposed larvae,

and the figures given may be deceptive. Also, the net

number of infected larvae produced (total number produced

minus number of infected larvae used in the inoculum) from

the test with the most inoculum was only about 1/3 of

that produced from a group exposed to 1/10 of the same

amount of inoculum.

Linley and Nielsen (1968a) conducted a series of

carefully described experiments on the transmission of

RMIV in the laboratory. The percentage of transmission

in these experiments was determined on the basis of the

number of larvae exposed, and exposure was not done in

the condition of extreme crowding that resulted in high

larval mortality for Woodard and Chapman (1968). However,









even with the modifications, their results generally

substantiated those of Woodard and Chapman (1968).

Matta and Lowe (1970) reported 2 separate infectivity

tests with known numbers of first stadium A. taeniorhynchus

larvae exposed to known amounts of inoculum and in which

the number of survivors was determined. Transmission rates

were 7.73 per cent and 15.6 per cent when calculated on

the basis of number of survivors, but only 2.67 per cent

and 5.95 per cent, respectively, when calculated on the

basis of number of larvae exposed, and larval mortality

was greater than 60 per cent in both tests. The increase

in virus produced (number of infected larvae produced,

minus number of infected larvae used as inoculum, divided

by number of infected larvae used as inoculum) was 167.5

per cent and 197.5 per cent in the respective experiments.

It was stated that the infectivity of the virus was not

significantly reduced by treatment with ether, but it

was not stated whether the test was performed as pre-

viously described by Andrewes and Horstmann (1949).

Experiments relative to the transmission of RMIV in

nature were described by Linley and Nielsen (1968b).

These experiments demonstrated that infection could be

acquired per os by healthy larvae exposed to virus from








either macerated or unmacerated larvae under field con-

ditions. Virus from macerated hosts retained its in-

fectivity in 10 per cent sea water for 3 days in the

laboratory, and then slowly attenuated over a period of

30 days. There was less retention of infectivity in

artesian water, and the virus was uninfective after 2

days on damp sod.

Transovarial transmission of RMIV was first observed

and substantiated by Woodard and Chapman (1968). Infected

larvae (never more than 2 per cent) were always found among

larvae reared from the eggs of survivors of experiments on

the infectivity of RMIV. Sterilization of all containers,

instruments and media did not reduce the level of trans-

mission. When late third or early fourth instar larvae

were exposed to virus, they showed no gross symptoms of

infection before or after becoming adults. However, in 3

experiments the larvae developing from eggs deposited by

these adults showed an average infection of almost 30 per

cent. Linley and Nielsen (1968a) excluded the possibility

of transovum transmission by surface sterilization of

the eggs. Additional support for a transovarial mechanism

was provided when Hall and Anthony (1971) found the virus

in both larval and adult ovaries.








All the progeny of 5 isolated, transmitting females

observed by Linley and Nielsen (1968a) and 3 observed by

Hall and Anthony (1971) were infected, suggesting an

all or none mechanism of transovarial transmission. The

5 transovarially transmitting females produced an average

of only 7.4 larvae while non-transmitting females of the

same generation produced an average of 26.7 larvae (Linley

and Nielsen, 1968a). This indicates the proportion of

transovarially transmitting females in a population is not

necessarily equal to the proportion of infected larvae

produced by that population. Evidence from 19 separate

tests indicated that virus infections were not carried

covertly from the second to the third generation (Linley

and Nielsen, 1968a).

Both Woodard and Chapman (1968) and Linley and

Nielsen (1968a) suggested that RMIV could be transmitted

to only a susceptible proportion of an exposed population.

They made no attempt to test that hypothesis nor did they

propose an explanation of why only a proportion of a

population would be susceptible. Woodard and Chapman

(1968) believed that the susceptible proportion of a

population remained fairly constant regardless of when the

larvae were exposed. This hypothesis was based on the








untenable assumption that the proportion of covertly in-

fected adults in a population was equal to the proportion

of infected larvae they produced.

Linley and Nielsen (1968b) proposed a tentative

account of the natural transmission of RMIV in A.

taeniorhynchus. They stated,

Transovarial transmission produces in-
fected larvae which die in the fourth
instar. These then provide possibly the
only, but certainly by far the most im-
portant, source of new infection, which
is acquired per os by the healthy larvae
when they feed on the diseased cadavers
prior to pupation. This in turn leads
to the presence of infected adults which
complete the cycle by depositing infected
eggs (p. 24).

Few observations have been made on the gross pathology

accompanying the development of infections with RMIV.

Chapman et al. (1960) observed that iridescence appeared

first in the thorax of late third or early fourth instar

larvae and then spread throughout the abdomen during the

fourth larval stadium. Most of the patently infected

larvae died before pupation. Matta and Lowe (1970) main-

tained that iridescence first appeared in the lateral

portions of one of the first four abdominal segments.

Linley and Nielsen (1968a) noted that larvae infected

transovarially appeared to move, feed and develop normally









until shortly before death, when they became sluggish and

ceased to feed. After death, the abdomen often contracted,

and some of the larvae remained suspended from the surface

of the water and were often cannibalized.

Clark et al. (1965) first studied the histopathology

of RMIV infections and stated that the virus appeared to

develop in cell cytoplasm of adipose tissue. Matta and

Lowe(1970), using darkfield microscopy and a staining

technique previously described by Matta and Lowe (1969),

observed the virus in fatbody and imaginal discs and noted

that the destruction of these tissues was usually complete

in patently infected larvae. In rare instances larvae sur-

vived, and in the pupae the fatbody had not been completely

destroyed. Using the electron microscope, Hall and

Anthony (1971) found the virus in tracheal epithelium and

epidermal tissues, in addition to fatbody and imaginal

discs, and to a lesser extent in hemocytes, foregut, nerve,

muscle, and in both larval and adult ovaries. Disruption

of metabolic processes in the larval fatbody was thought

to be the cause of death. Virus was never found in midgut

or hindgut epithelium or in malpighian tubules.

In attempts to locate the site of entry of RMIV into

host tissue Hall and Anthony (1971) found virus particles








in the esophageal region of the foregut epithelium after

larvae were exposed to suspensions of the virus. It is

unlikely that the virus passed through the cuticular lining

in this region of the gut, and it was not stated how long

the larvae had been exposed to the virus suspension.

Stoltz and Summers (1971) presented the hypothesis that

virus entered the epithelium in the extreme anterior region

of the midgut. This hypothesis was based, in part, on tne

observation that intact virus particles were not found more

than 100 p distal to the foregut invagination into the

midgut. The peritrophic membrane has been considered an

effective barrier to intact virions, but both Hall and

Anthony (1971) and Stoltz and Summers (1971) expressed the

possibility that subviral components of nucleic acid or.

nucleoprotein might pass through the membrane.

The present dissertation further elucidates the re-

lationships between RMIV and A. taeniorhynchus. Techniques

for mass production and purification of large amounts of

RMIV in minimal amounts of time were essential to several

of these efforts. Tinsley and Harrap (1972) stated at

the Second International Congress for Virology that one of

the main difficulties in research with MIV was its low

virulence and the resulting lack of sufficient quantities









of the virus obtained from laboratory hosts.

priority was placed on the development of met

produce large (gram) quantities of purified I

dardized management of the host was regarded

to the mass production of the virus. Severa:

ment methods involving different feeding rec

use of different rearing containers were atte

the most successful of these has been descril

ments were conducted to determine the optimui

inoculum necessary and the optimum age at wh

larvae to assure maximum net virus production:

tion of RMIV and the effect of infection wit

the weight of the host were quantitated. Th

of infectivity of RMIV resulting from purifi

studied to determine if it could be attribut

to any particular one of the procedures in p

Several experiments were conducted to elucid

tative aspects of vertical transmission with

nation and to determine if it was possible f









failures to achieve high rates of transmission by j

exposures. The site of entry of RMIV into host ti:

was sought by fluorescent antibody and autoradiogr

observations with the light microscope and by exam

of ultrathin sections of exposed first instar larvy

the electron microscope. The development of overt

fections with RMIV in larvae exposed in the second

stadium was studied by fluorescent antibody stainii

by autoradiography. The development of covert inf

with RMIV in larvae, pupae and adults, exposed as

instar larvae, was studied by fluorescent antibody

The number of structural polypeptides composing RM

determined and their molecular weights estimated b

polyacrylamide gel disc electrophoresis. Attempts

made to isolate these polypeptides by hydroxylapat

chromatography.















METHODS AND MATERIALS


Production of Virus


Routine Virus Production
Procedures

The initial source of RMIV used in these studies

was live, infected, fourth stadium larvae of A. taenior-

hynchus mosquitoes collected from a small saltmarsh on

the southwest side of Atsena Otie Key, an island near

the town of Cedar Key, Florida. All mosquito eggs used

to produce larvae for routine virus production were acquired

from the mosquito colony of the U. S. Department of Agri-

culture Insects Affecting Man and Animals Research Labora-

tory, at Gainesville, Florida. The colony procedures used

for mosquito production are being described for publica-

tion (Ford and Green, in prep.).

Eggs were obtained from the colony on damp spnagnum moss

in pans covered with aluminum foil. They could be stored

at room temperature for up to 3 months without unacceptable

loss of vigor. Periodic examinations of larvae produced

in the colony provided assurance that there were no latent









virus infections present. Tap water was used to rinse

the eggs from the moss into a round enamel pan, using

a screen to collect the moss and debris. The water was

swirled rapidly to accumulate the eggs in the center of

the pan, and they were then pipetted into graduated conical

bottom centrifuge tubes, 1 cm3 per tube. Each tube of

eggs was poured into an enamel hatching pan containing 2

liters of 0.05 M NaCl made with distilled water. The eggs

were again accumulated by swirling, and an infusion of

200 mg of live brewer's yeast was added to each pan. The

eggs were allowed to hatch for 1 hr after which the un-

hatched eggs were removed. One cm3 of eggs provided about

20,000 larvae in each pan. These were allowed to develop

for 24 hr in the yeast infusion.

The progression of mosquito life was measured by days,

or 24 hr periods, beginning at hatching, regardless of the

time of day this occurred. No effort was made to coordinate

any treatment with moulting or ecdysis, but it is pertinent

that passage through each of the first 3 stadia required

about 24 hr with adequate food and space (Nayar, 1967).

All water temperatures were maintained at 270C throughout

rearing.

Twenty-four-hr-old larvae were poured onto an organdy








cloth screen, washed with tap water, and placed in a small

volume of 0.05 M saline. Eight oz waxed-paper cups were

used as containers to expose the larvae to virus. Twenty-

five hundred larvae were counted into 1 cup containing

200 ml of 0.05 M saline, and this cup was then used as a

visual standard to pipette an estimated 2500 larvae into

each exposure container to be used. Inoculum was prepared

by triturating fresh, RMIV infected fourth stadium larvae

in a Ten Broeck type tissue grinder until their head cap-

sules were completely destroyed. It was quantitated as

larval equivalents (LEQ), 1 LEQ being the virus from 1

patently infected larva, whether freshly triturated or at

some stage in virus purification. The inoculum was filtered

through an organdy screen before being pipetted into the

exposure containers. Routine exposures were for 24 hr

without additional food.

Rearing containers, prepared several hours before

needed, were white plastic trays,1 56.7 x 44.7 x 8.0 cm,

containing 7.5 liters of 0.05 M saline made with table



panel Control Corp., Detroit, Michigan.









salt and tap water. After exposure, the larvae and

inocula were poured into the rearing trays. One gram

of high protein, low fat content hog food supplemental

(ground fine enough to pass through a screen of 50 meshes

per inch) was added as a thoroughly wetted slurry to

each tray. The larvae were fed an additional 2 grams

of food 48 and 72 hr after being placed in the rearing

trays. With this schedule, no aeration was necessary,

and no fouling of the water occurred. Occasionally, a

few pupae appeared late in the fifth day of larval life,

but pupation usually began late in the sixth day after the

eggs hatched.

The collection of infected larvae began when the first

pupae were observed in the trays. The contents of the

trays were poured through a screen of 30 meshes per inch

to retain the larvae. The trays were washed immediately

with cold tap water and a scrub cloth to remove all old

infusion and detritus. Soap was never used, and no attempt

was made to sterilize the trays, although they were allowed

to dry thoroughly before being used again. The larvae



ISoutheast Hog Supplement (40% protein), Purina,
St. Louis, Missouri.









were placed in a black photographic tray containing ice

water to immobilize them. The infected larvae, which

were conspicuously iridescent against the black back-

ground, were removed with a bulbed pipette and held in

cold 0.05 M Tris-HCl buffer, pH 7.0.


Determination of Optimum
Quantity of RMIV Inoculum

Eggs were hatched and larvae allowed to develop for

24 hr as described above. Six experimental groups of

larvae, each with replicates of 2500 larvae, were exposed

for 24 hr to 5, 10, 25, 50, 75, and 100 LEQ of inoculum

per group, respectively. Controls were exposed to an equal

number of triturated uninfected larvae. Groups exposed

to an inoculum of only 5 LEQ were given a small amount of

food. At the end of the exposure period, all groups were

examined for larval mortality, and the larvae were trans-

ferred to rearing trays and maintained as described above.

Feeding of groups that experienced mortality during

exposure was reduced to avoid contaminating the rearing

containers with excessive food. Infected larvae from these

treatments were harvested and counted late in the fourth

stadium. Only larvae showing visible signs of RMIV in-

fection were considered infected. Net production of virus,









quantitated as LEQ, was determined. A qualitative judge-

ment of mortality was made, since only a low level of

mortality could be tolerated in any virus production system

to be used routinely. The experiment was repeated 3

times in successive weeks to be certain the system was

replicable.


Determination of the Ootimum
Larval Age for Exposure to RMIV

Six experimental groups, composed of 2 replicates of

2500 larvae each, were exposed for 24 hr to 50 LEQ of

RMIV inoculum during days 1 through 6 of larval life,

respectively. Exposure of group 1 was made immediately

after hatching, and exposure of group 2 was made during the

second day of larval life as described for routine virus

production. Larvae of groups 3 through 6 were placed in

rearing trays when 24 hr old, fed and held by routine

methods until the respective groups were old enough to

expose. After exposure, all larvae were carefully rinsed

with tap water to remove external inoculum and returned

to rearing trays. Groups that had been exposed were

screened and infected larvae collected and counted at 12

hr intervals beginning early in the fourth day of larval

life and continuing through day 7, when pupation was almost








complete. Net production of virus, quantitated as LEQ,

was determined from 2 experiments conducted at an interval

of 1 month.


Purification of RMIV

All virus purification procedures were performed at

00 40C in 0.05 M Tris HC1 buffer, pH 7.0. Virus

infected larvae were triturated in a Ten Broeck type tissue

grinder until their head capsules were completely destroyed.

The triturant was washed through organdy cloth into a

beaker, and the volume was adjusted to 50 ml per 1000 larvae.

The beaker was covered and the contents stirred with a

magnetic stirrer for 12 24 hr to further fragment the

tissues. The triturant was then placed in a separatory

funnel and brought to a 20 per cent (v/v) mixture with

cold ethyl ether. The mixture was shaken vigorously for

5 min, placed in an explosion proof refrigerator, and

allowed to separate for 6 hr. The buffer layer was removed

and re-extracted with ether. The first ether layer was

washed with the original volume of buffer and allowed

to separate an additional 6 hr. These extractions were

effective in removing much of the debris from the virus

suspension. The final buffer layers were combined in a









beaker and placed in an ice bath. The residual ether was

removed by stirring the mixture gently (to avoid foaming)

for 12 24 hr with a magnetic stirrer under a ventilated

hood, while passing a gentle stream of air over the top

of the virus suspension. Following ether extraction,

the virus was pelleted by centrifugation in 50 ml round

bottom centrifuge tubes at 10,000 g for 40 min, in an

International Equipment Company (IEC) PR-6 refrigerated

centrifuge equipped with a high speed accessory attachment.

The supernatant buffer was discarded, 10 ml of new buffer

were added to each tube, and the pellets were allowed to

soften overnight before being resuspended by vortexing.

After resuspension, the volume was adjusted to 80 ml per

1000 LEQ being processed, and the virus was subjected to

4 cycles of differential centrifugation. The low speed

cycle was 15 min at 1000 g, and the high speed cycle was

40 min at 10,000 g. The low speed pellets of the first

3 cycles were scavenged by resuspending them to 25 per

cent of their original volume, combining them, and

recentrifuging them for 20 min at 1500 g. The virus in

the supernatant buffer was pelleted by a high speed run.

All high speed pellets were allowed to soften in buffer

for at least 6 hr before being resuspended by vortexing.








Virus in the high speed pellets from the scavenging runs

was returned to the main bulk of virus before the succeed-

ing low speed run. The final low speed pellet was dis-

carded.

The concentration of resuspended virus from tne final

high speed run was estimated spectrophotometrically and

adjusted to approximately 20 mg/ml with a Beckman DU-2

spectrophotometer, and the absorption curve at 700 nm

(Matta, 1970). Sucrose density gradients were prepared by

successively layering 2.5 ml aliquots of 55 per cent

(w/v), 44 per cent, 30 per cent, and 15 per cent sucrose

in 13 ml, 1.4 x 9.7 cm centrifuge tubes. The gradients

were allowed to form naturally in cold for at least 12 hr

before use, and gradients not used within 48 hr of prepara-

tion were discarded. The virus suspension was diluted

1:1 with 10 per cent sucrose, and 2 ml of this material

were layered on the gradients and centrifuged at 20,000 g

for 20 min in an IEC B-60 ultracentrifuge equipped with a

type SB-283 head. After centrifugation, the virus was

seen as a distinctive band about two-thirds of the way

down the tube, with a diffuse layer of top component slightly

above it. Initially, a small pellet of iridescent material

was seen on the bottom of the tubes after sucrose gradient









centrifugation, but later results showed that proper care

in disrupting and resuspending the final high speed

pellet from differential centrifugation prevented its forma-

tion. The virus band, but not the top component, was

removed with a needle and syringe. The virus suspension

was diluted 1:1 with distilled water and pelleted in a

Beckman Model L preparative ultracentrifuge equipped with

an SW-39 head. The pellets were resuspended and washed

twice with distilled water to remove the residual sucrose.

The final pellets were resuspended in a minimum volume of

distilled water, quantitated spectrophotometrically, ad-

justed to a concentration of 15 mg/ml, and lyophylized

with a Virtis lyophylizer. The lyophylized virus (15 mg/

tube) was stored at -300C.

Virus purified by this procedure reacted vigorously

with its homologous antiserum formed in rabbits, but not

with normal rabbit serum, in tube precipitin tests. It

did not react with antiserum against acetone extracted

A. taeniorhynchus tissue in tube precipitin tests, by agar

gel diffusion tests, or by immunoelectrophoresis in agarose.

It appeared homogeneous in the electron microscope.

Matta (1970) found that virus purified by a similar but

less rigorous method produced a single sedimentation boun-

dary in an analytical ultracentrifuge equipped with









Schlieren optics.


Quantification of RMIV
Production

Fifteen groups of 2500 larvae each were exposed to

an inoculum of 50 LEQ per group and reared to the advanced

fourth stadium as described for routine virus production.

Infected larvae were collected and counted, and the total

number was divided into 2 equal groups. A third group of

larvae, showing no overt symptoms of RMIV infection and

equal in number to 1/2 the total number of infected larvae,

was also collected. The virus from one group of the in-

fected larvae was purified by methods described above and

quantitated spectrophotometrically. The other group of

infected larvae and the group of apparently uninfected

larvae were lyophylized with a Virtis lyophylizer until

weight loss stopped. The weight of both groups was deter-

mined on a Mettler H-5 balance. From these data the

following results were determined: (1) the per cent of

transmission acquired by routine virus production pro-

cedures, (2) the quantity of purified virus produced per

infected larva, (3) the weights of infected and uninfected

larvae from the same rearing set, and (4) the per cent of

larval dry weight attributed to the virus.









Attenuation of Infectivity
of RMIV During Purification

Transmission studies with purified, lyophylized virus

indicated that there was lower transmission with this

material than when other preparation were used. Experi-

ments were conducted to determine if the loss of infectivity

occurred at any particular stage of the virus purification

procedure.

Six experimental classes were established, each com-

posed of 5 groups of 200 larvae. Twenty-four-hr-old larvae

were exposed for 24 hr to virus which had been purified

through various stages of purification as follows: (1)

fresh, triturated infected larvae; (2) buffer extracted

virus; (3) ether extracted virus; (4) virus subjected to

4 cycles of differential centrifugation; (5) virus which

had been subjected to sucrose density gradient centri-

fugation; and (6) virus which had been purified and

lyophylized.

Larvae in classes 1 and 2 were exposed to 5 LEQ of

inoculum per group. Larvae in classes 3 and 4 were exposed

to 10 LEQ per group. Larvae in classes 5 and 6 were

exposed to 20 LEQ per group. The use of increasing amounts

of inoculum was necessary to assure infections in all

classes, because preliminary tests showed that exposure of









classes 1 and 2 to the quantities of inoculum necessary

to produce infections in classes 5 and 6 resulted in high

mortality during exposure. A small amount of food was

provided larvae in classes 3 through 6 during exposure,

and for each group 200 larvae were reared in rectangular,

white enamel pans containing 1 liter of 0.05 M NaCl.

The larvae were examined for RMIV infection in the late

fourth stadium, and per cent transmission was determined

for each class. The experiment was replicated each week

for 4 weeks.



Transmission of RMIV


per Cent of Larvae Infected
with RMIV by Transovarial
Transmission

Experiments were conducted to determine the per cent

of infected Fl generation larvae produced by adults that

had survived exposure to RMIV at different times during

their larval life. The surviving adults from the 6 experi-

mental groups in the experiment "Determination of the Optimum

Age for Exposure to RMIV" were maintained in an adult

rearing room at 800C and 70 per cent relative humidity.

Cotton pads soaked in 5 per cent sucrose were continually

available as a food source, and shaved guinea pigs provided









blood meals on consecutive days. Five days after emergence,

16 oz cups, containing loosely packed, damp sphagnum moss

were offered as an oviposition medium. The moss was

left in the cages for 5 days, then covered with aluminum

foil, and the collected eggs were allowed to develop an

additional 10 days. The eggs then were hatched and reared

for 24 hr as described for routine virus production. Two

trays of 2500 larvae from each group were reared to the

advanced fourth stadium. They were examined for RMIV

infection, and the per cent transmission in each group

was determined. The experiment was conducted twice in

successive months.


Transovarial Transmission
Through Isolated Females

Larvae were reared to 4 days of age by procedures

described above, at which time groups of 2500 were exposed

to 50 LEQ of inoculum for 24 hr. The larvae were rinsed

thoroughly after exposure to remove inoculum, placed in a

rearing tray and checked periodically for evidence of

overtly infected larvae until pupation was completed. The

pupae were placed in a cage to emerge, and the adults

were fed as described above. Five days after emergence

was completed, 100 blooded females were taken from the









group and isolated in 35 ml glass vials containing damp

sphagnum moss as an oviposition medium. Boiled raisins

were placed on the screened tops of the -ials for food and

as a source of liquid. The females were left in the vials

for 1 week, then removed. The vial tops were taped to

prevent desiccation of the eggs, and the eggs were allowed

to develop an additional 2 weeks. The moss from each

vial was then placed on a screen and washed. Egg batches

from vials in which oviposition occurred were placed indivi-

dually in rectangular, white enamel pans with 1 liter of

0.05 M NaC1. Larvae hatching in the pans were fed and

allowed to develop to the advanced fourth stadium, counted

and examined for RMIV infection. Determined from these

data were: (1) the per cent of isolated blooded females

that oviposited, (2) the per cent of the egg batches pro-

duced which hatched, (3) the per cent of the egg batches

which hatched that contained infected larvae, (4) the per

cent of infected larvae from each egg batch producing

infected larvae, and (5) the number of larvae produced

by transmitting and by non-transmitting females.









Sex Specificity of Transovarial
Transmission of RMIV

Four rearing trays of 2500 A. taeniorhynchus larvae

each were allowed to develop to 5 days of age. The larvae

in 2 of the trays were exposed for 24 hr in 16 oz cups

to 100 LEQ of fresh inoculum per cup of 2500 larvae.

After exposure, the larvae were rinsed thoroughly and re-

turned to rearing trays. The 2 untreated trays were main-

tained on a regular rearing schedule. Pupae from all 4

trays were collected and put into cages for emergence.

At 6 hr intervals during a 48 hr emergence period, the

adults were immobilized in a cold room and separated both

according to sex, and whether or not they had been exposed

to virus. When emergence was complete the adults in

each cage were again immobilized and divided into 2 equal

groups and recombined to result in the following experi-

mental classes:

Class 1: Unexposed Males X Unexposed Females

Class 2: Unexposed Males X Exposed Females

Class 3: Exposed Males X Unexposed Females

Class 4: Exposed Males X Exposed Females

The adults were fed as previously described. Eggs

were again collected by the standard method and allowed








to develop for 10 days after oviposition. They were then

hatched, and 2 groups of 2500 larvae from each class were

allowed to develop to advanced fourth stadium, when they

were examined for evidence of RMIV infection. The per

cent of transovarial transmission of RMIV in each class

was determined.


Transmission of RMIV by
Integument Puncture

An attempt was made to transmit RMIV by integument

puncture to determine if rates of transmission higher than

those acquired per os could be achieved. Late third instar

larvae (early in their fourth day of larval life) were

rinsed thoroughly with tap water and placed in a suspension

of 1 mg/ml of purified RMIV in a petri dish placed in a

tray of shaved ice. The depth of the virus suspension

was sufficient to cover the larvae, and when they were

immobilized, they were pinched with a pair of fine tipped

forceps, prepared as described by Clark (1966), and trans-

ferred to a covered tray of 0.15 M NaC1. After 24 hr,

the survivors were transferred to a tray of clear 0.05

M NaC1. To extend their development time, they were

maintained at 240C rather than 270C, as in routine rearing,

and only a small amount of food was provided. Beginning








48 hr after inoculation, the larvae were checked at 12 hr

intervals for infection with RMIV.


Production of Antiserum to RMIV and
to A. taeniorhynchus Antigens


Antibodies to both RMIV and A. taeniorhynchus antigens

were produced in rabbits. Baseline normal sera were

acquired by cardiac puncture of nonimmunized rabbits.

Formation of antibody to RMIV was stimulated by 4 weekly

injections of virus into each rabbit used. Five mg of

purified RMIV was suspended in 1 ml of normal saline,

emulsified with 1 mg of Freund's complete adjuvant, and

1 ml of this emulsion was injected intramuscularly into

each hip. Weekly bleedings (35-40 ml) by cardiac puncture

began I wk after the fourth injection. Injection of

antigen without adjuvant was continued after the fourth

week, with the injections being given immediately after

blood was withdrawn. The blood was allowed to clot in 50

ml centrifuge tubes for 1 hr at room temperature. The

clots were then ringed with a sterile applicator stick and

allowed to retract for 24 hr at a temperature of 40C. The



1Difco Laboratories, Detroit, Michigan.








tubes were centrifuged at 5,000 g for 20 min, and the serum

was collected with sterile pipetts and stored without

glycerine at -300C. The presence of antibody to RMIV

was determined by the tube precipitin test.

Powdered tissue of A. taeniorhynchus was prepared

by triturating healthy fourth stadium larvae in a Ten

Broeck type tissue grinder, containing acetone, in a dry

ice-acetone bath. The tissue was extracted with acetone

and the powdered tissue collected by filtration at -300C.

The powdered tissue was air dried, and water soluble

proteins to be used as antigens were solubilized with

phosphate buffered saline, pH 7.0, by stirring overnight

at 4C. Insoluble matter was removed by centrifugation

at 15,000 g for 30 min and discarded. The antigen was

sterilized by passing it through an 0.45 u Millipore

filter. The concentration of protein was determined by

the Lowry method (Lowry et al., 1951), using bovine serum

albumin fraction V1 as a standard. Antibody to this

antigen was produced in a rabbit by injecting 10 mg of

antigen suspended in Fruend's complete adjuvant weekly

for 4 weeks. Only 1 collection of 35 ml of blood



lSigma Chemical Company, St. Louis, Missouri.








was made by cardian puncture before the rabbit expired.

The antiserum was prepared and stored as described

above. Presence of antibody was determined by the tube

precipitin test.


Purification of Gamma Globulin


Methods used to purify gamma globulin from anti-

RMIV antiserum were partially those of Goldman (1968) and

partially those of Kawamura (1969). All procedures were

performed at 00 40C. Antiserum was diluted 1:1 with

sodium phosphate buffered saline, pH 7.0. Saturated enzyme

grade ammonium sulfate,1 (H4)2SO4, was adjusted to pH

7.0 with 0.1 N NaOH. The globulins were precipitated twice

with half-saturated (NH4)2SO4 and the euglobulins were

precipitated twice with 1/3 saturated (NH4)2SO4. The

precipitates were resolubilized in distilled water to the

original undiluted serum volume, except for the final

precipitates, which were dissolved in 1/2 the original

serum volume. The residual (NH4)2SO4 was removed by

dialysis against 0.15 M NaCd for 24 hr with stirring and

frequent changes of saline. The globulins were then

dialized against 0.005 M phosphate buffer, pH 8.0. The



1Sigma Chemical Company, St. Louis, Missouri.








protein concentration was determined by the Lowry method

(Lowry et al., 1951) and adjusted to 40 mg/ml by

pressure filtration in an Amicon pressure filtration

device equipped with a PM-10 membrane. A DEAE cellulose2

column was prepared with a dry weight of DEAE cellulose

equal to 20 times the actual quantity of proteins to be

separated. A column with a height : diameter ratio of

more than 10:1 was used. The column was equilibrated and

eluted with 0.005 M phosphate buffer, pH 8.0, in which

both alpha and beta globulins, but not gamma globulin,

adsorbed to the column. The column was eluted with a flow

rate of 10 ml/hr. The gamma globulin from the column was

quantitated spectrophotometrically, using an extinction

coefficient of 1.24, and the concentration was adjusted

to 20 mg/ml. The gamma globulin was stored at 40C for

a short time before further processing.


Conjuqation of Anti-RMIV Antibody with
Fluoresceine Isothiocyanate (FITC)


Conjugation was performed by the method of Clark and

Shepard (1963) as described by Goldman (1968). The



1Amicon Corporation, Lexington, Massachusetts.

2Sigma Chemical Company, St. Louis, Missouri. 0.89
meg/g.








concentration of the antibody solution was adjusted to

20 mg/ml and the pH was adjusted to 9.5 with 0.5 M

carbonate-bicarbonate buffer, pH 9.5, containing 1 mg

FITC1 per ml of immune globulin G (IgG) solution.

A G-25 Sphadex2 desalting column was prepared and

equilibrated with 0.0175 M phosphate buffer, pH 6.3. The

solution of conjugated antibody and free dye was added to

the column, and the column was washed with the equilibrating

buffer. The conjugated antibody was eluted after the

void volume, while the free dye was retained on the column.

The volume of the labeled antibody was adjusted to the

pre-labeling IgG volume with an Amicon pressure filtration

device equipped with a PM-10 membrane. The labeled IgG

was then dialyzed overnight against a large volume of

0.0175 M phosphate buffer, pH 6.3.

At this point the antibody solution consisted of

molecules of unlabeled antibody and antibody molecules to

which were conjugated 1 or more FITC molecules.



lBaltimore Biological Laboratory, Baltimore, Maryland.

Sigma Chemical Company, St. Louis, Missouri.








Kawamura (1969) stated that the unlabeled antibody should

be removed, because it would compete with labeled anti-

body for binding sites. Also, antibody molecules with

more than 2 FITC molecules attached should be removed,

because they had a high net negative charge and tended

to combine non-specifically with tissue. To this end, the

labeled antibody solution was subjected to chromatography

on a DEAE cellulose column equilibrated with 0.0175 M

phosphate buffer, pH 6.3. In this buffer unlabeled anti-

body would not absorb to the column, and it could be

eluted from the column with the equilibrating buffer.

The labeled antibody was eluted from the column, according

to the number of FITC molecules attached, by eluting the

column with a flow rate of 10 ml/hr of 0.0175 M phosphate

buffer, pH 6.3, containing successively 0.1, 0.2, and

0.3 M NaC1.

The fluoresceine : protein (F : P) ratio of the

conjugate was determined by the method of Holborow and

Johnson (1967), using the formula:


Concentration of FITC (ug/ml) =

Absorption492 Absorption320 x Dilution
2
0.2








Protein concentration was then determined by the Lowry

method (Lowry et al., 1951), and the F : P ratio was

determined by the formula:


F : P = 0.41 x pg FITC/ml
mg/ml protein


Fractions with F : P ratios of 0.36 to 1.79 were

pooled, with a resulting composite F : P ratio of 0.76.

The labeled antibody was dialyzed exhaustively against

phosphate buffered saline, pH 7.2. The concentration was

adjusted to 8.33 mg/ml, and the conjugate was lyophylized

in lots of 10 mg and 15 mg/tube and stored at -300C.

For evaluation, the labeled antibody was reconstituted

to its pre-lyophylization volume with distilled water,

diluted with phosphate buffered saline, pH 7.2, to a con-

centration of 3 mg/ml, and clarified by centrifugation at

15,000 g for 10 min. Dilutions of 2 mg/ml, 1 mg/ml, 0.5

mg/ml and 0.25 mg/ml were prepared to determine the

optimum concentration to use for staining. Unexposed and

known infected larvae as test specimens were embedded

together in Paraplast,1 the blocks were sectioned at a



1Scientific products, Evanston, Illinois.








thickness of 8 u, and the sections were deparaffinized in

tertiary butanol at 400C. The specimens were hydrated

through an ethanol series to distilled water and washed

in phosphate buffered saline before being incubated in a

humidified chamber with dilutions of the conjugated

antibody. Following exposure to the antibody, they were

given 4 5 min washes in phosphate buffered saline and

coverslips were mounted over the specimens with a

media of 1 per cent Tris /-Tris hydroxymethyll) amino-

menthane_7 in 9 parts glycerine and 1 part water, with

the pH adjusted to 9.9 with 0.1 N NaOH (Mrenova and

Albrecht, 1966). The specimens were examined and photo-

graphed with a Zeiss Ultrophot II microscope, using dark

field illumination. The light source was an Osram HBO-200

Mercury vapor lamp. The primary filter used was the

Zeiss UG-1/3mm, and the secondary filter admitted all

wave lengths of light greater than 410 nm. Dilutions of

the labeled antibody as low as 0.5 mg/ml were found

to stain foci of virus infections, and subsequent studies

utilized a concentration of 1 mg/ml. The specificity of

the stain was indicated by its failure to stain sections

of uninfected larvae mounted alternately with sections

of infected larvae, on the same slide. The staining








reaction of the labeled antibody was inhibited by treating

sections of infected larvae with unlabeled anti-RMIV anti-

serum prior to treatment with the labeled antibody. Treat-

ing sections of infected larvae with antiserum to A.

taeniorhynchus did not inhibit the staining reaction.


Pilot Project on Autoradiography of 3H-Methyl
Thymidine Uptake and Distribution in
Larval A. taeniorhynchus


No literature was available on certain parameters

essential to an autoradiographic study of the development

of RMIV infection in A. taeniorhynchus larvae. A pilot

project was conducted with uninfected larvae to determine:

(1) the level of radioactivity necessary in an aqueous

medium to assure uptake of 3H-methyl thymidine into larvae

in quantities detectable autoradiographically, (2) the

length of time larvae could be exposed to this activity

without destroying the resolution of the technique by

translocation of the tritiated methyl group by metabolism,

and (3) the optimum length of time to expose a nuclear

track emulsion to sections containing the tritiated

thymidine, before developing them.

Dilutions of 3H-methyl thymidine,l with a specific



lInternational Chemical and Nuclear Corporation, Irvine,
California.








activity of 17.4 Curies/millimole (Ci/mM) and a concentra-

tion of 1 millicurie/mM (mCi/mM), were made with 0.05 M

NaCl to provide solutions of 2.5 microcuries/Mi (pCi/ml),

5pCi/ml, 10 pCi/ml, and 25 pCi/ml. Ten ml of each con-

centration were placed in each of 4 aluminum weighing cups

and a small amount of brewer's yeast infusion added. Ten

late third stadium larvae of A. taeniorhynchus were placed

in each cup. At intervals of 30 min, 1 hr, 2 hr, and 4 hr

after initiation of exposure, the larvae in 1 of the 4

cups of each concentration were removed, rinsed thoroughly,

and placed for 30 min in 3 oz cups containing a yeast

infusion. The post-exposure feeding was intended to flush

unabsorbed label from the gut to minimize the nonspecific

distribution of the label by the microtome knife during

sectioning of the specimens. Following exposure to label,

the larvae were washed thoroughly in a dilute solution of

Tween-80, rinsed in distilled water, fixed in carnoy's

fixative for 4 hr at room temperature, dehydrated through

absolute ethanoi, cleared in tertiary butanol and embedded,

two larvae per block, in paraplast. Serial sections of the

larvae from the 16 experimental groups were cut at a

thickness of 8 p on a Leitz microtome and placed on

gelatin cooled slides. The slides were divided into 5








sets, each set containing the serial sections of two

larvae from each of the experimental groups. The slides

were deparaffinized in xylene, hydrated through descending

concentrations of ethanol to distilled water and allowed

to dry. All subsequent dipping, packaging and developing

operations were performed in total darkness. The slides

were emulsion coated by dipping them in Kodak NTB-2 nuclear

track emulsion1 that had been melted and held in a constant

temperature water bath at 450C. Control blank slides, to

determine background graination, and unlabeled sections,

to check for possible nonspecific tissue activity, were

also coated, and all slides were allowed to dry for 2 hr

in a near vertical position. After drying, the slides were

sealed in slide boxes containing a drying agent, wrapped

in several layers of aluminum foil, and placed in a

refrigerator at 40C. At intervals of 2, 3, 5, 7 and 10

weeks after dipping, 1 set of the slides was developed

in Kodak Dektol for 1.5 min at 18C, dipped for 30 sec

into Kodak Indicator Stopbath, fixed for 10 min in Kodak

Rapid Fix (diluted 1:1), and washed in running water for

15 min (Rogers, 1967). After rinsing in distilled water,



1Eastman Kodak Company, Rochester, New York.








the slides were stained in methyl green-puronine (Pease,

1953) as recommended by Thurston and Joftes (1963). After

staining, the slides were dehydrated in tertiary butanol,

cleared in tolune and coverslips were mounted with a

synthetic resinous mounting medium.

When all sets of the autoradiograms had been processed,

they were examined at 500 x (oil) magnification with an

AO-Spencer microscope. Background was rated on the con-

trol slides and also on each experimental slide, by examin-

ing areas of the slide distant from sections, as light

( 5 grains/100 u2), moderate (5-10 grains/100 u2) or heavy

( 10 grains/100 u2). The quality of the stain reaction

was noted, and the control sections which contained no

radioisotope were examined for evidence of nonspecific

activity. The following tissues were examined for evidence

of Label uptake: epidermis, fatbody, foregut epithelium,

midgut epithelium, gastric caecae, imaginal discs, hindgut

epithelium, malpighian tubules, tracheal epithelium and

gonads. Casual observation was made of incorporation into

nerve and muscle tissue. Labeling over the midgut contents

was observed to see if the gut had been swept clean by

post exposure feeding. Incorporation of label into the

cells of each tissue, as indicated by supranuclear








grairntion, :,as rated semiquantitatively by code as

follows:

Frequency of labeled cells (nuclei)

1 = -1/10 of cells labeled

2 = 1/10 1/2 of cells labeled

3 = >1/2 of cells labeled

Intensity of activity in labeled cells (nuclei)

1 ,= z 25 grains per nucleus

2 = 25-50 grains per nucleus

3 = 50-100 grains per nucleus

4 = ?100 grains per nucleus

Only the effect of the various exposure concentrations

and durations on the labeling of the midgut epithelium

cells was considered in deciding the concentration of label

to use in subsequent experiments. These cells were ob-

served as standards, because (a) they were uniformly large,

(b) the nuclei occupied less than 1/2 the area of the cells

in cross-sections, making it easy to discern successful

localization of the isotope within the nucleus, and (c)

because they had good staining properties.








Production of 3H-Methyl Thymidine
Labeled RMIV


A. taeniorhynchus larvae were hatched and reared for

24 hr, and 2000 of them were exposed to RMIV as described

for routine virus production. The larvae were reared

to advanced third stadium and then transferred to a pan

containing a liter of 0.05 M NaCd, 10 mCi of 3H-methyl

thymidine (specific activity of 66 Ci/mnM), and a small

amount of food. The larvae were exposed to the tritiated

thymidine for 24 hr. At the end of exposure 128 con-

spicuously infected fourth stadium larvae were harvested,

as were 10 larvae which displayed no evidence of infection.

The uninfected larvae and four of the infected larvae were

prepared for autoradiography as described above. The

virus in the remaining 124 infected larvae was purified by

methods previously described and quantitated spectro-

photometrically. Five mg of labeled virus were recovered

and diluted to 50 mg. An 0.2 ml sample gave 1200 disin-

tegrations per min (dpm) above background when counted in

a Packard Tri-carb Liquid Scintillation Counter, indicat-

ing a total radioactivity of 0.14 pCi for the purified

virus. The virus was used immediately in autoradiographic

study of the site of penetration of RMIV into host tissue.









Site of Entry of RMIV into
Host Tissue


Light Microscope Studies

Autoradiographv.--Eggs of A. taeniorhynchus were

hatched and the larvae allowed to develop for 24 hr by

standard procedures. Two thousand larvae were exposed to

5 mg of RVIV, labeled with 3H-methyl thymidine as

described before, with a total radioactivity of 0.14

pCi. At intervals of 1/4, 1/2, 1, 2, 4, 8, and 12 hr,

125 of the larvae were withdrawn from the exposure medium,

rinsed thoroughly with tap water, and fixed in Carnoy's

fixative for 4 hr at room temperature. The specimens

were processed histologically, dipped in Kodak NTB-2

nuclear track emulsion, developed after 2 weeks' exposure

and stained as previously described. They were examined

at magnifications of 500x (oil) and 1000x (oil) in an

attempt to detect a site of entry of the labeled virus

through the gut wall.

Exposed specimens that were not processed for auto-

radiography were reared to the advanced fourth stadium,

when they were examined to determine the per cent infection

with RMIV.








Fluorescent antibody.--Eggs of A. taeniorhynchus

were hatched, the larvae were allowed to develop for 24

hr, and 2500 larvae were exposed to an inoculum of 50 LEQ

of RMIV as described for routine virus production. At

intervals of 1/4, 1/2, 1, 2, 4, 8, 12, and 24 hr after the

initiation of exposure to virus, samples of 125 larvae

were removed from the exposure container. They were rinsed

thoroughly, fixed in Carnoy's fixative for 4 hr at room

temperature, dehydrated through absolute ethanol, cleared

in tertiary butanol, and embedded in Paraplast. Sections,

processed as before, were incubated for 45 min with

1 mg/ml conjugated anti-RMIV antibody and examined as

for other studies for evidence of a specific site of entry

of RMIV into host tissue.

Larvae not collected for processing were allowed to

develop to the advanced fourth stadium when they were

examined to determine per cent infection with RMIV.


Electron Microscope Studies

Two hundred freshly hatched A. taeniorhynchus larvae

were placed in a 10 cm diameter petri dish of distilled

water containing an inoculum of 10 freshly triturated

RMIV infected fourth stadium larvae. Samples of exposed

larvae were removed from the exposure medium at intervals








of 5, 10, 15, 20, and 30 min, and also at 1 1/2, 2, 3,

4, and 5 hr after the initiation of exposure. Head

capsules and air tubes were excised in glutaraldehyde,

and the larvae were fixed for 4 hr at room temperature

in 4 per cent glutaraldehyde in 0.1 M cacodylate buffer,

pH 7.2. They were washed in cacodylate buffer over night,

post fixed for 1 hr in 1 per cent osmium tetroxide in

cacodylate buffer, dehydrated through ascending concen-

trations of ethanol, cleared in propylene oxide, and

embedded in epon-araldite (Mollenhauer, 1964). Silver to

light gold sections, approximately 60 nm thick, were cut

on a Sorvall Porter-Blum MT-2 ultramicrotome with glass

knives, and the sections were taken up on carbon or

Formvar coated grids. The sections were stained with

saturated uranyl acetate and lead citrate (Venable and

Coggeshall, 1965) and examined and photographed with a

Hitachi 125 E electron microscope using accelerating

voltages of 50 or 75 KV. Thick sections were also cut

and examined by phase contract microscopy as an aid to

orientation.









Development of RMIV Infections


Autoradiography

Eggs of A. taeniorhynchus were hatched, and the larvae

were allowed to develop for 48 hr in the hatching container,

by which time they had reached the second larval stadium.

Two groups, each with 2500 larvae, were exposed for 24 hr

to 100 LEQ of RMIV inoculum and then transferred to rearing

containers. Twelve hours after the termination of virus

exposure, and at 24 hr intervals thereafter, 125 of the

exposed larvae were removed from the rearing container and

placed for 2 hr into 125 ml of 0.05 M NaCl containing 20 pCi/ml

of 3H-methyl thymidine with a specific activity of 13 Ci/mM.

A small amount of food was provided to encourage the intake

and passage of material through the gut. At the termination

of exposure to tritiated thymidine, the larvae were

thoroughly rinsed and placed in a suspension of food for

30 min. The larvae were processed histologically, and

autoradiography and staining were performed as has been

described. Exposure of this emulsion was for a period of

10 days. The activity of the exposure medium was increased

to 20 pCi/ml, despite the results of the pilot project,

and the emulsion exposure time was decreased in an attempt









to shorten the experiment. Autoradiograms were examined

at magnifications'500x (oil) and 1000x (oil) to determine

the tissue in which infection could be detected by this

technique and to determine how soon after the exposure

infections in these tissues could be detected.

Larvae not collected for study were reared to the

advanced fourth stadium and examined to determine the

per cent infection with RMIV.


Fluorescent Antibody

Overt infections.--Eggs of A. taeniorhynchus were

hatched, the larvae were reared for 24 hr, and 2500 of the

larvae were exposed to 50 LEQ of RMIV inoculum as described

for routine virus production. At the termination of ex-

posure, the larvae were transferred to a rearing container.

At that time and at 24 hr intervals thereafter, samples

of 125 larvae were collected, processed histologically,

and stained with fluorescent antibody, 1 mg/ml, as described

above.

The exposed specimens were examined to determine in

which tissues infection could be detected and at what

time interval after initiation of exposure infections

could be detected by this method.









Larvae not collected for this study were reared to

the advanced fourth stadium and examined to determine the

per cent of infection with RMIV.


Covert infections.--Specimens for this study were

collected from the exposed mosquitoes in the experiment,

"Sex Specificity of Transovarial Transmission of RMIV."

Larvae were exposed to 100 LEQ of RMIV inoculum per group

of 2500 larvae during the fifth day of larval life.

Specimens for this study were collected as larvae at the

end of the sixth day of larval life (48 hr after exposure

was initiated), as pupae at the end of the eighth day of

life, just prior to eclosion (72 hr after exposure was

initiated), and as adult females prior to bleeding (120

hr after exposure was initiated). Known transovarially

transmitting females, surviving the experiment "Transmission

of RMIV through Isolated Females," were also examined.

The specimens were processed histologically, stained with

fluorescent antibody to RMIV (1 mg protein/ml) and examined

as described above to determine which tissues were infected

and at what time interval after the initiation of exposure

infection could be detected, with special attention paid

to reproductive tissues. The amount of transovarial trans-

mission occurring through the experimental group, exclusive








of the known infected females, was reflected in the trans-

mission through class 4 of the experiment "Sex Specificity

of Transovarial Transmission of RMIV."


Number and Molecular Weights of RMIV
Structural Polypeptides


Analytical sodium dodecyl sulfate (SDS)--polyacrylamide

gel disc electrophoresis was performed on SDS--2-

mercaptoethanol disrupted samples of highly purified RMIV

by the methods of Maizel (1971). Purified RMIV was sub-

jected to sucrose gradient centrifugation 1 or 2 additional

times, washed 3 times with distilled water to remove

residual sucrose, and quantitated spectrophotometrically

(Matta, 1970). The concentration was adjusted to 10 mg

(protein)/ml in distilled water. The virus was structurally

disrupted and the proteins denatured to polypeptides by

heating the virus to 1000C for 1 min in 1 per cent SDS1

and 0.1 per cent 2-mercaptoethanol.2 Samples of 100 ug,

200 ug, 300 ug and 400 ug of protein in 250 ul volume of

12 per cent sucrose were overlaid onto 10 x 0.6 cm resolving



1Fisher Scientific Company, Fairlawn, New Jersey.

2Matheson, Coleman and Bell, Cincinnati, Ohio.









gels of 10 per cent or 13 per cent acrylamidel with

2.5 x 0.6 cm stacking gels of 3 per cent acrylamide. Both

resolving gels and stacking gels contained 0.1 per cent

SDS. Bromphenol blue was added to the samples as a

tracking dye. Simultaneously, 50 ug samples of proteins

of known molecular weight,2 also treated with SDS and 2-

mercaptoethanol, were run in identical gels. A current of

2mA per tube was applied until the tracking dye had migrated

0.5 cm into the resolving gel, and then 4mA per tube were

applied until the tracking dye approached the bottom of the

resolving gels. The gels were cut at the dye front when re-

moved from their tubes. They were fixed and stained in 0.2 per

cent Coomassie Brilliant Blue R 2503 in 50 per cent methanol

and 7 per cent acetic acid for 12 hr and destined in repeated

changes of 5 per cent methanol in 7 per cent acetic acid.

The gels were stored in stoppered test tubes containing 7

per cent acetic acid. The stained bands of viral poly-

peptides were counted, and the relative migration of both



ISigma Chemical Company, St. Louis, Missouri.

2Schwarz/Mann, Orangeburg, New York.

3Sigma Chemical Company, St. Louis, Missouri.








viral polypeptides and polypeptides of known molecular

weight was determined in relationship to the dye front.

Curves were drawn, relating to relative migration of the

standard polypeptides in both 10 per cent and 13 per cent

resolving gels, to the Logl0 of their molecular weights.

The molecular weights of the viral polypeptides were then

estimated from these curves.


Isolation of RMIV Structural Polypeptides
by Hydroxylapatite Chromatography


Hydroxylapatite chromatography techniques described

by Moss and Rosemblum (1972) were utilized with RMIV.

Hydroxylapatite (Bio-Gel HT) was washed repeatedly with

0.01 M sodium phosphate, pH 6.4, 0.1 per cent (W/V) SDS,

and 1.0 mM dithiothreitol2 (DTT). Columns, 2.5 x 10 CM,

were poured over a layer of fine grade Sephardex G-502

or over filter paper discs. Twenty to 40 mg samples of

RMIV, purified as described above, were made to 5 ml in

1 per cent SDS, 0.1 per cent 2-mercaptoethanol and placed

in a boiling water bath for 2 min. The denatured samples



1Bio-Rod Laboratories, Richmond, California.

2Sigma Chemical Company, St. Louis, Missouri.









were then diluted to a concentration of 1 mg/ml with 0.01

M sodium phosphate, pH 6.4, 0.1 per cent SDS and placed on

the columns. They were washed into the columns with 2

column volumes of the 0.01 M phosphate-SDS-DTT solution and

eluted with a linear gradient formed by a 2-chambered

gradient former, containing 200 ml of 0.1 M sodium phosphate,

pH 6.4, 0.1 per cent SDS, 1 mM DTT in the starting chamber and

200 ml of 0.7M sodium phosphate, pH 6.4, 0.1 per cent SDS, 1

mM DTT in the trailing chamber. A flow rate of about

5 ml/hr was maintained by gravity. Three ml fractions were

collected in a refrigerated Buchler fraction collector.

The 0.D280 of the fractions was determined in a Beckman

DU-2 spectrophotometer, and curves were drawn relating

fraction number to 0.D.280















RESULTS AND DISCUSSION


Production of RMIV


Determination of Optimum
Quantity of RMIV Inoculum

For purposes of this dissertation, the optimum

quantity of RMIV inoculum for use in the routine produc-

tion of RMIV was defined as that quantity which resulted

in the maximum net production of virus without causing

significant larval mortality during exposure. The combined

results of 3 experiments designed to determine that

quantity are shown in Table 1. An inoculum of 50 LEQ per

exposure cup of 2500 larvae was found to be the optimum

inoculum for use in the virus production system described

and was used in routine virus production.

A graph of these data (Figure 1) indicates that the

per cent transmission of RMIV increased in proportion to

the quantity of inoculum used until inocula in excess of

50 LEQ/2500 larvae were used. Above this point, high

larval mortality during exposure obscured the relationship

between per cent transmission and quantity of inoculum.












Table 1

Net Virus Production Resulting from 24 Hr Exposure
of Day Old A. taeniorhynchus
Larvae to RMIV




Inoculum (LEQ) Per 2500 Larvae
5 10 25 50 75 100

Gross Number of Infected
Larvae Per 15,000 Exposed 234 317 758 1174 863 131

Per Cent Transmission 1.56 2.1 5.1 7.2 5.7 0.8

Net Virus Production
(LEQ) Per 15,000 Exposed 219 287 863 927 638 0

Mortality During Very
Exposure Low Low Low Low High High






64




7





6





r 5
0





4
S4-












2-





1







10 20 30 40 50 60 70 80 90 100

Inoculum (LEQ) per 2500 larvae

Fig. 1. Per Cent Transmission of RMIV in A. taeniorhynchus
Larvae Exposed to Increasing Amounts of Inoculum.









Mortality during exposure in the control groups

paralleled that in the experimental groups, indicating

that the high mortality in experimental groups exposed

to 75 and 100 LEQ of inoculum was not due to a toxic

effect of the virus, as experienced by Bellett and Mercer

(1964) in studies on the multiplication of SIV in cell

cultures.

No infection was observed among the control larvae.


Determination of Optimum
Larval Age for Exposure
to RMIV

The combined results of two experiments designed to

determine the effect of the age of A. taeniorhynchus larvae

at exposure to RMIV on the net production of RMIV are

shown in Table 2. Maximum net production of RMIV resulted

from exposures initiated when the larvae were 24 hr old.

These results corroborate those of Woodard and Chapman

(1968).


Quantification of RMIV
Production

Thirty-eight hundred infected, advanced fourth stadium

larvae were collected from 37,500 larvae exposed to RMIV

and maintained as described for routine virus production.












Table 2

RMIV Production Resulting from 24 Hr Exposure
of A. taeniorhynchus Larvae of Different
Ages to 50 LEQ of Inoculum


Day of Larval Life Exposure
Was Effected
1 2 3 4 5 6

Gross Number of Infected
Larvae per 10,000 Exposed 1033 1084 702 329 0 0

Per Cent Transmission 10.33 10.84 7.02 3.20 0 0

Net Virus Production
(LEQ) per 10,000 Larvae
Exposed 833 884 502 129 0 0








This represented a transmission rate of 10.13 per cent.

Nineteen hundred infected larvae and an equal number of

uninfected larvae from the same experiment had dry weights

of 1.19698 grams and 1.39290 grams, respectively. This

represented a mean dry weight of 630 pg and 733 pg for

infected and uninfected larvae, respectively. From the

additional 1900 infected larvae harvested, 112 mg of RMIV

were purified. This was a recovery of 58.9 pg of purified

virus per infected larva and showed that 9.35 per cent of

the dry weight of an average infected, advanced fourth

stadium larva was composed of virus.

The host production system and the virus purification

procedures described here resulted in a maximum production

by 1 person of 940 mg of purified virus in 1 week and have

approached that quantity several other times. Therefore,

it is possible for 1 person, with an adequate mosquito

colony, setting 80 trays (200,000) of exposed larvae per

week, to conveniently produce and purify 1 net gram of

virus per week. Virus produced in 1 week can be purified

the subsequent week while a new generation of infected

hosts are produced. Although this quantity of virus may

not be adequate for extensive biological control field

testing, it certainly is adequate for numerous laboratory

studies.









Attenuation of Infectivity
of RMIV Durinq purification

An attenuation of the infectivity of RMIV during

purification is apparent from the data shown in Table 3.

Almost 70 times as many infected larvae per LEQ of inoculum

were produced by freshly triturated inoculum as by an

inoculum of purified virus that had been lyophylized. This

attenuation may have resulted from both quantitative loss

of virus during purification and from some damage to virus

particles during processing that reduced the ability to

enter a host cell or the ability to establish active infec-

tions. The proportion of attenuation attributable to any

one of these causes cannot be determined from the data.

The greatest attenuation appeared to occur following

ether extraction and sucrose gradient centrifugation. This

was surprising, since RMIV was shown to be insensitive to

ether by Matta and Lowe (1970) and because sucrose density

gradient centrifugation is commonly used in virus purifi-

cation because it is a relatively mild technique. The

greater relative loss of infectivity during these stages of

purification was possibly a reflection either of the use of

increased quantities of inoculum in those experimental groups

or a reflection of a non-linear relationship between trans-

mission and the quantity of inoculum used. Despite the






















progressive Stage Quant
of Purification (LEQ)

Trituration

Buffer Extraction

Ether Extraction

Differential Centrifugatior

Sucrose Gradient
Centrifugation

Lyophylization


Table 3

Attenuation of Infectivity Resulting
from purification of RMIV Through
the Stage Indicated

Infected Larvae Per Cent
tity of Inoculum Produced Per Trans-
Per 4,000 Larvae 4,000 Exposed mission

100 337 8.4

100 189 4.7

200 109 2.7

S200 69 1.7


400 39 1.0

400 22 0.5


Infected Larvae
Produced Per LEQ
of Inoculum

3.37

1.89

0.55

0.35


0.10

0.05








ambiguity of the data, it was apparent that the attenuation

detected was not caused by any single step in purification,

but occurred throughout the whole procedure.


Transmission of RMIV


Per Cent of Larvae Infected
with RMIV by Transovarial
Transmission

A relationship was found between the age of the parent

generation when exposed to RMIV and the frequency of in-

fections transmitted transovarially. As shown in Figure 2,

little or no transovarial transmission into progeny occurred

when the parent generation was exposed to virus during early

larval life. The amount of transovarial transmission in-

creased as the age of the parent generation at exposure

increased, up to a point, and then decreased. These data

suggest that, when sufficient time for development of

infections in larvae is available, overt infections with

RMIV result. No definite explanation can be given for

the decrease in transovarial transmission through parents

exposed during the sixth day of larval life. It is possible

that some host physiological change associated with pupa-

tion interrupts some infections before reproductive tissues

become infected. This possibility will be discussed further

in later sections.













(3.54%)


(2.24%)


(1.99%)


(0%/) (0.17%)

1 2 3
Day of larval life during
tion was exposed to RMIV


4 5 6
which the parent genera-


Fig. 2. per Cent of RMIV Infection Among Progeny of Mos-
quitoes Exposed to RMIV as Larvae of Different Ages.

Data from 2 experiments were combined, and a total
of 10,000 larvae were in each experimental group.








Transmission of RMIV
Through Isolated Females

Eighty-three of 100 isolated,blooded females, exposed

to RMIV during the fifth day of larval life, oviposited.

Of these, eggs of 67 of the females (81%) hatched, and 9

groups (13.4%) contained infected larvae. Initially,

some of the larvae in 4 of the infected groups displayed

no gross infection early in the fourth stadium. This

suggested that there were exceptions to the apparent all-or-

none mechanism of transovarial transmission observed by

Woodard and Chapman (1968) and by Hall and Anthony (1971).

However, the uninfected larvae were isolated, and without

exception they developed overt infections and died before

pupating. The 9 groups of infected larvae consisted of

from 13 to 104 larvae, with a mean of 63.3 per group,

compared to the uninfected groups, which ranged from 1 to

156 larvae with a mean of 94.2 per group. From the total

of 6,053 larval progeny observed from the isolated females,

only 591 (9.8%) were infected, although 13.4 per cent (9

of 67) of the females were known to have been infected.

These data suggested that the per cent of infection among

larvae produced in transovarial transmission experiments

was not an accurate reflection of the per cent of infection









occurring among the females that produced them. In the

experiment described, the per cent of known infected

females among the isolated females exceeded the per cent

of overt infections developing among the progeny of the

experimental group by a factor of 1.37 (13.4% + 9.8% = 1.37).

Although this factor was acquired from limited data, it

will be used in subsequent discussions to estimate the

per cent of infected (transmitting) females in transovarial

transmission experiments from the per cent of infection

among the progeny of the experimental group.


Sex Specificity of
Transovarial Transmission

Males of A. taeniorhvnchus apparently play no part in

the transovarial transmission of RMIV (Table 4). When males

were exposed to RMIV as 4 day-old larvae and mated to unex-

posed females, none of 5,000 progeny were infected. Also,

per cent of infection among progeny of exposed females was

not diminished by mating them to unexposed males rather than

to exposed males.

As will be discussed in later sections, overt infec-

tions appeared as often among male larvae as among female

larvae when mixed groups were exposed in early instars.

Since RMIV is a cytoplasmic virus, the apparent inability












Table 4

Transovarial Transmission of RMIV by Reciprocal
Mating of Exposed and Unexposed Male and
Female A. taeniorhynchus


Per Cent Infection Among Progeny
Mating (5,000 Larvae Per Class)


Unexposed Males x Unexposed Females 0

Unexposed Males x Exposed Females 4.5

Exposed Males x Unexposed Females 0

Exposed Males x Exposed Females 4.8









of males to transmit the virus may be related to the very

small quantity of cytoplasm occurring in sperm.


Total of Overt and
Covert Transmission

The total amount of RMIV infection acquired by a

group of exposed larvae was defined as the sum of overt

infections (identified by the characteristic iridescence

of infected larvae and pupae) and covert infections. Overt

infection with RMIV were easy to quantitate, and since

they are fatal almost without exception, they play no

part in transovarial transmission. Little is known of

covert infections with RMIV, since at present the only

evidence for their existence is the transovarial transmission

resulting from a portion of them. The proportion of covert

infections which do not result in transmission is unknown.

Also, it is not known whether covert infections can result

in transovarial transmission into only 1 of numerous

ovipositions by a single female. Nevertheless, an attempt

was made to determine the total transmission of RMIV to

groups of larvae exposed to the virus at different ages.

This was done to test the hypothesis (Woodard and Chapman,

1968) that about the same per cent of infections occurred

whether the larvae were exposed when young (resulting in








overt infections) or when older (resulting in transovarial

transmission).

The following assumptions were necessary: (1) males

play no significant part in vertical transmission of the

virus, although they become infected with the same fre-

quency as females; (2) the per cent of covertly infected

females is about 1.37 times the per cent of infected

larvae among the progeny of the exposed generation; and (3)

the proportion of covertly infected females which transmit

the virus through their first egg batch is not influenced

by the age of the larvae when infection is acquired.

Support for the first 2 assumptions has been offered. The

latter is not unreasonable, considering that overt infections

are manifested 3 or 4 days after initiation of exposures

in larvae, and that adults are usually a week to 10 days

old before they oviposit. Therefore, infections should

have had time to develop regardless of when the larvae

acquired infection. Overt infections occurring in the

experiment "Determination of Optimum Larval Age for

Exposure to RMIV" were related to covert infections, re-

flected by transovarial transmission, in the experiment

"Per cent of Larvae Infected with RMIV by Transovarial

Transmission," because the ova for the latter experiment













Table 5

Total of Overt and Covert Transmission
of RMIV to Larvae Exposed at
Different Ages


per Cent Total
Day During Which Per Cent Overt Covert Trans-
Larvae Were Exposed Transmission Transmission* mission**

1 10.33 0 10.33

2 10.84 0.23 11.07

3 7.02 0.62 7.64

4 3.29 3.07 6.36

5 0 4.85 4.85

6 0 2.73 2.73


*Covert transmission was calculated as 1.37 times per cent
of infection among progeny of the exposed generation.

**These data represent observation of 10,000 larvae per
class.









were produced by the survivors of the former. Both experi-

ments were conducted twice at an interval of 1 month, and

combined data are given in Table 5. These data suggest

that older larvae are less susceptible to infection with

RMIV than younger larvae and that larvae are more sus-

ceptible to RMIV during the second day of larval life than

at any other time.


Transmission of RMIV
by Integument Puncture

Attempts to transmit RMIV to A. taeniorhvnchus larvae

by integument puncture, as described by Clark (1966), were

unsuccessful. Clark reported up to 70 per cent transmission

of various microbial mosquito pathogens by this method.

One hundred and seventy-five of 269 third instar larvae

survived the exposure and were fourth instar larvae or

pupae 5 days after exposure. Pupation was delayed by

a poor diet and by maintaining the larvae at 24C rather

than at 270C. These conditions attempted to prolong

development and may have interfered with the development

of infection. Also, there is no assurance that virus ever

entered the host, so the data are inconclusive.








Pilot Project on Autoradioaraphy of 3H-Methyl
Thymidine Uptake and Distribution in
Larval A. taeniorhvnchus


Autoradiography detected uptake of 3H-methyl thymidine

into the nuclei of cells of third instar A. taeniorhynchus

larvae in the following tissues: epidermis, fatbody,

foregut epithelium, midgut epithelium, gastric caecae,

imaginal buds, hindgut epithelium, malpighian tubules,

tracheal epithelium and the gonads. Uptake was greatest

in the malpighian tubules, hindgut epithelium, and gastric

caecae, and only slightly less in midgut epithelium,

maginal buds, and fatbody. Less uptake was detected in

epidermis, foregut epithelium, tracheal epithelium and gonadal

tissue. Uptake into nerve and muscle cells was rarely

detected. Exposure to 2.5 pCi/ml, for up to 4 hr, resulted

in labeling too sparse to be useful, but exposure to

25 jCi/ml for even 1/2 hr overlabeled many tissues. A

2 hr treatment of 10 pCi/ml labeled the cells of most

tissues consistently and adequately. On the basis of the

response of midgut epithelium (used as a representative

tissue) this treatment was selected as ideal for a study

of the development of RMIV infections in host tissue. In

Figure 3 is shown the sharply circumscribed location of























































Fig. 3. Malpighian Tubule Cell with Labeled Nucleus
(100x) .
(N Nucleus)









graination over a labeled nucleus. The specific activity

of the labeled thymidine used in this pilot study was

17 Ci/mM and exposures of 2 weeks were adequate for

specimens exposed to the accepted treatment of 10 pCi/ml

for 2 hr. Grains over the cells of active tissues

increased in number as duration of exposure of the emulsion

increased, but background graination also increased rapidly

and reduced the resolution of the technique.

Examination of control materials indicated that back-

ground graination intrinsic to the emulsion was low enough

not to reduce the resolution of the technique. A small

amount of very fine graination occurred over the anterior

thoracic fatbody of all specimens, whether exposed to

label or not, and could not be explained. The staining

reaction of methyl green-pryonine was very good in unin-

fected tissue. DNA in the nuclei stained a pale blue,

and the graination was easy to detect. RNA stained a pale

pink in the cytoplasm and a bright pink in the nucleoles,

which was very helpful in detecting the nuclei of small

cells.

Feeding larvae to flush unabsorbed tritiated thymidine

from the gut lumen after exposure was very helpful. In

the few specimens that did not feed during the flushing








period, graination was very heavy over the gut contents.

It was observed that some of the radioactive thymidine

was dispersed from the gut into surrounding areas by the

microtome blade during sectioning. An interesting

phenomenon was observed in larvae that had fed actively

during the flushing period. As shown in Figure 4, a pocket

of tritiated thymidine remained in the otherwise cleanly

flushed gut. The residual pocket was ring-shaped and

located in the anterior part of the midgut at the level of

the caudal extremity of the foregut invagination into the

midgut. This possibly represented an "eddy" in the flow

of material through the gut, and its possible significance

is discussed later with studies on the site of entry of

RMIV into host tissue.


Site of Entry of RMIV
Into Host Tissue


Light Microscope Studies

Autoradiography.--The site of entry of RMIV into host

tissue could not be detected on autoradiograms of serial

sections of 700 early instar larvae of A. taeniorhynchus

exposed to purified RMIV that had been labeled with

3H-methyl thymidine. Three and one-tenth per cent (30/973)





















































Fig. 4. Anterior Midgut Region of Larva Showing Residual
Deposits of Tritiated Thyvidine Peripheral to
Caudal Extremity of Foregut Invagination into
Midgut (100x).
(I Foregut invagination: C Gastric caecae)








of the survivors of exposure to the purified, labeled

virus developed infections with RMIV, when they were

reared to the fourth stadium. That was about the same

per cent transmission as achieved with purified, unlabeled

virus in the study "Attenuation of Infectivity of RMIV

During Purification," suggesting that labeling did not

reduce the infectivity of the virus. Small foci of grains

were seen scattered in the emulsion over the midgut

contents of larvae that had been exposed to the labeled

virus, indicating that labeled virus was consumed and

that it could be detected autoradiographically. However,

these foci of grains were rare. Only 0.14 uCi of activity

were detectable in the entire virus inoculum (5 mg) by

liquid scintillation counting. Autoradiograms of sections

of 4 of the infected larvae in which the inoculum was

produced were examined. These revealed that label

incorporation into foci of virus, identified in the

cytoplasm of infected cells by their staining reaction with

methyl green, was much less than into the nuclei of the

infected cells (Figure 5). The evidence suggests that the

virus inoculum was not heavily labeled. This could have

occurred if most of the viral DNA had been produced before

the infected larvae were exposed to tritiated thymidine.

















































Fig. 5. RMIV Infected Fatbody Cell in A. taeniorhynchus
Larva with Graination Over Cytoplasmic Deposits
of Tritiated Thymidine Labeled Viral DNA (1000x).








If this is true, it might account for failure to observe

exit of the virus from the gut lumen into host tissue by

autoradiography.


Fluorescent antibody.--The site of entry of RMIV into

host tissue was not detected by examination of fluorescent

antibody stained serial sections from 800 early instar

larvae exposed to an inoculum of triturated, infected

hosts. Infection with RMIV developed in 16.8 per cent (191/

1131) of the survivors of the exposure, when they were

reared to the fourth stadium. Studies to be discussed

below suggested that RMIV entered host tissue as isolated

virions which did not become aggregated in cells of the mid-

gut epithelium. Neither these virions nor the fluorescence

from them by attached labeled antibody could have been

detected with the light microscope because their size is

below the resolution of the equipment used.


Electron Microscooe Studies

A probable site of entry of RMIV into host tissue was

found by examination of tissues with the electron micro-

scope. Numerous virus particles with the size and appearance

of RMIV were found in midgut epithelium of first instar

A. taeniorhynchus larvae killed and fixed 5 hr after









initiation of exposure to the virus. The infected

epithelial cell shown in Figures 6 and 7 is at the level

of the foregut invagination into the midgut. This was de-

termined by the relative positions of the infected cell,

the peritrophic membrane and the cuticular lining of the

foregut invagination. The larvae examined were from a

colony known to be free of RMIV infection prior to exposure.

A possible mechanism of entry of RMIV into host tissue

is as follows. Some of the virus particles entering the

midgut are swept into the eddys forming a ring around the

caudal extremity of the foregut invagination. (See Figure

4 and related text.) Some of these virus particles are

pressed against the peritrophic membrane (Figures 8 and 9),

emerging between the foregut invagination and the midgut

wall (Figures 6 and 7). Where perforations occur in the

peritrophic membrane due to faulty construction or to

trauma, virus particles invade the space between the

peritrophic membrane and the midgut epithelium. Virus

particles and other objects of equal or larger size were

seen between the peritrophic membrane and the midgut wall

(Figures 8, 10 and 11). Virus particles enter the midgut

cells, perhaps passing into and down the microvilli

(Figures 10 and 11), or perhaps crossing the cell membrane

in an area where the microvilli are disarrayed or damaged




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