ETH Library. Doctoral Thesis. Author(s): Drolet, Jacques. Publication date: Permanent link:

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The nuclear and the cytoplasmic granulosis viruses of Adoxophyes orana F.v.R. (Lep., Tortricidae)

separation, biochemical characterization, pathogenesis and infectivity

Doctoral Thesis Author(s):

Drolet, Jacques Publication date:

1989

Permanent link:

https://doi.org/10.3929/ethz-a-000510338 Rights / license:

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Diss. ETH No. 8899

The nuclear and the cytoplasmic granulosis ^{viruses of} Adoxophyes

^orana

^F.v.R. (Lep., Tortricidae): Separation,

bioehemical characterization, pathogenesis ^and infectivity

Dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZÜRICH

for the

degree

^of

Doctor of Natural Sciences

presented by Jacques

^Drolet

M.Sc. Universite

Laval,

^Quebec born

July 3,1956

Citizen ofCanada

accepted

^on the recommendation of Prof. Dr. G.

Benz,

^examiner Prof. Dr. P.

Lüthy,

co-examiner

1989

CatE

49

Summary

The electron

microscopical

^{studies of Schmid etal.}

(1983)

indicated that the

granulosis

^of

Adoxophyes

^orana

F.v.R.,

^found in the Valais

(Switzer¬

land),

^{followed two different}

paths

^{indifferentfat}

body

^cells:

(i)

^the

classic form of

granulosis

^virus

development

^{where the}

synthesis

^{of the}

virus occurs in the nucleus and later in the "nuclearfield"

(nuclear type) (Wäger

^and

Benz, 1971)

^and

(ii)

^an

atypical

form where the nucleus does not show any transformation and the

virogenesis

^{occurs in the}

cytoplasm

(cytoplasmic type) (Benz, 1979). By infecting

^Ist instar larvaewith the contentsof individual fat

body

^{cells of infected 5th instar}

larvae,

^two

types

of virus have been

separated.

^The

Separation

^{was stable forat leastthree}

generations.

^The

pathogenesis

^{wasfollowed at}the tissue Ievel with

phase-contrast microscopy.

The method enables the Observation of a

relatively high

number of cells which is essential to

study

^the

dynamics

^of

the infection in the cell

population

^{at the}tissue Ievel.

Thecharacterization of thetwo

types

^{was made with}

SDS-polyacrilamide gel electrophoresis

and restriction endonuclease

analysis.

^The

banding

^of

the

proteins

^{showed a}difference in 5

major proteins

between thetwo

types

of virus

(SDS-PAGE).

^{Thisdifferencewas notfound at}the DNA Ievel when studied with restriction endonuclease

analysis.

^Some

hypotheses

^are

proposed

^to

explain

^this

discrepancy.

The rates ofvirus

production

^{in the fat}

body

^were 2.6 and 1.9 for the

cytoplasmic

^{and the nuclear}

type respectively

when the infection wasdone with the natural mixture of thetwo

types,

^{i.e. the}

proportion

^{found in dead}

larvae infected with thetwo

types. However,

^{the rates ofvirus}

production

foreach virus

type

^{was inferiorto these}values when the infection was

done with an artificial mixture of the two

types (cytoplasmic

^{: nuclear= 1 :}

1).

^{This shows the}

importance

^{of the}

proportion

^{of each virus}

type

^{in the}

Suspension

^{used for}

infecting

the larvae and the

advantage,

^atleastatthe

tissue

Ievel,

^{ofthe ratio of thetwo virus}

types

found in the natural mixture.

The effect of the

temperature

^{on the}

development

of the infectionwas

different foreach

type: high temperatures

^{reduce the rate of}

development

^of

the nuclear

type

^{while low}

temperatures

^{have the same effecton the}

cytoplasmic type.

Infection with

high

concentrations

(10.8

ⁱⁿ

log10)

^{gave a lower}

^proportion

of infected fat

body

^{cells at}the end of the larval

development

^than

infection with lower

(6.8

ⁱⁿ

log-jg)

concentrations. This may indicate ^a

negative

^{effect of}

high

virus doses on the virus

yield

per larva.

Bioassays

^{weremade to}compare the

infectivity

^{of thetwo}

types

^{of virus at} different

temperatures.

^{In orderto} diminish the

variability

^{of the}

virus-host

system,

^the

following

^factorswere standardized: the

storage time,

the time ofextraction of the virus after

infection,

^theestimation of the virus

concentration,

and the volume

ingested

per larva. The natural

mixture had a

LC50

^{of 3.03 x}

¹⁰⁷

^{GV/ml at25°C.}

^Compared

^{to this}

^value,

^the

cytoplasmic type

of virus has

only

^{0.63 times this}

potency

^and the nuclear

type

^0.54times

(%2

^«

7.0,

^{a ¦}

0.05).

^The

persistence

^ofthe mixture in the natural

population

^{can therefore be}

explained.

The effect ofthe

temperature

^{on the}

LC50

^{is not}

^simply explained by

^the

51

rate ofvirus

production. High

^{and low}

temperatures

^{actas stress factors}

on the insects in a process

parallel

^{to the} infection which may kill the

insect, although

^{the infection rate itself} is hindered when

compared

^{to the}

one observed for middle

temperatures.

The

LT^qS

^ofthetwo

^types

^{ofvirus were} similar but influenced

by

^the

temperature: high temperatures

^{reduced the}

LT5Q.

The effect of the

infection with

high

concentrationswas to raise the LT50

compared

^{to the}

infection with low

concentrations,

ⁱⁿ

agreement

with the results obtained at thetissue Ievel.

Resume

L' etude de

microscopie electronique

^executee par Schmid etal.

(1983)

^a

indique

que la

granulöse d'Adoxophyes

^{orana F. v.}

R.,

^{trouveeau Valais}

(Suisse),

^suivaitdeux

types

^de

developpement

differents dansdifferentes cellules du corps gras:

(i)

^{la forme}

classique

^de

developpement

^{de la}

granulöse

^{oü la}

synthese

^{du virusdebute}dans le noyau ^{et se}

poursuit

^{dans le}

champ

^nucleaire

(type-nucleique) (Wäger

^et

Benz, 1971)

^and

(ii)

^{une forme}

atypique

^oü le noyau ^ne revele aucune transformationet oü la

virogenese

^se

produit

^{dans le}

cytoplasme (type-cytoplasmique).

^{Les deux}

types

^{de virus}

ontete separes ^en infectant des larves de

premier

^{Stade aveclecontenu de}

cellules individuelles

provenantde

^larves infectees de

5e

Stade. La

Separation

^{s'est revelee} stable pour^au moins trois passages. ^La

Pathogenese

a et§ suivie au niveau du tissue ä l'aided'un

microscope

^{äcontrastede}

phase.

Cette methode

permet

l'observation d'un nombre relativement

grand

decellulesce

qui

^{est necessaire ä} l'etude de la

dynamique

^de l'infection dans la

population

de cellules des corps gras de l'insecte.

La caracterisation des deux

types

^{a ete}effectuee par

Pelectrophorese

^des

proteines

^sur

gel

^de

polyacrilamide (SDS- PAGE)

^etpar ^une

analyse

^{de l'ADN}

par enzymes de restriction. Une difference ^au niveau de

cinq proteines

majeures

^{estobservee entre les deux}

types

^{de virus.}

Cependant, Panalyse

^de

l'ADN n'a_{pu mettre} en evidence aucunedifference entre les deux

types

^de virus. Des

hypotheses

^sontdiscutees pour

expliquer

^{ces resultats.}

Les taux de

production

^{du virus}dans le tissu sont de 2.6 et 1.9 pour le

type-cytoplasmique

^etle

type-nucleique respectivement, lorsque

l'infection esteffectueeavec une

Suspension

^du

melange

natural des deux

types

^de

53

virus. Le

melange

^{naturel est la}

proportion

^{trouveedans une larve morte}

infectee par les deux

types

^{de virus.}

Cependant,

^lestauxde

production

^de

chaque type

de virus sont inferieurs äcesvaleurs

lorsque

l'infection est effectuee avecune

Suspension

^du

melange

artificiel des deux

types

^{de virus}

(cytoplasmique

^:

nucleique

^{= 1}

:1).

^{Ceci montre}

rimportance

^{de la}

proportion

de

chaque

^{virus dans la}

Suspension

utilisee pour l'infection ^des larves et

l'avantage,

^{toutau moins au} niveau de l'infection du

tissu,

^{de la}

proportion

trouvee dans le

melange

^naturel.

L'effetde la

temperature

^{sur le}

developpement

^de l'infection estdifferent pour

chaque type

^devirus: les hautes

temperatures

reduisent le taux de

developpement

^du

type nucleique

alors que les basses

temperatures

^{ont le}

meme effet surle

type cytoplasmique.

Lors d'infections ädoses elevees

(10.8

^en

log^),

^la

^proportion

de cellules

du corps gras infecteesä la fin du

developpement

^{larvaire est inferieure ä}

celle observee lors d'infections ädoses

plus

^basses

(6.8

^en

log-jn)

^ce

^qui

demontre un effet

negatif

des hautes concentrationssur la

production

^de

virus par larve.

Des bioessaisont ete effectues pour comparer la virulence des deux

types

de virusä des

temperatures

differentes. Dans le but de reduire la variabilite inherente au

complexe virus-höte,

^{les Clementssuivants ont ete}

uniformises: le

temps

^de

conservation,

^le

_temps

d'extraction du virus apres

infection,

Testimation de la concentration du virusetlevolume de

Suspension ingere

par larve. Le

melange

^{naturel a une} concentration lethale moyenne

(CL50)

^{de 3.03 x}

¹⁰⁷

^{GV/ml ä}

25°C. Compare

^äcette

^valeur,

^le

type

cytoplasmique

^{a0.63 fois cette} infectivite et le

type nucleique

^0.54fois

(statistiquement

^{differentavec}

^*2

⁼

7.0,

^{a =}

0.05).

L'infectivite

plus

^elevee

du

melange

^naturel

explique

^{done en}

partie

^la

persistence

^{des deux}

types

^de

virus sur le terrain.

Le taux de

developpement

^du virus dans le corps gras

n'explique

pas ä lui seul l'effet de la

temperature

^{sur la}

CL^q.

Des facteurs de stress

agissant

sur

l'insecte,

^{tel hautes etbasses}

temperatures, agissent

^aussi

paralleles

^ä

l'infection

elle-meme,

^ce

qui

^se manifeste par ^une mortalite larvaire accentuee, ^en

depit

d'un Processus d'infection ralentie par

rapport

^äcelui observe pour les

temperatures

intermediaires.

Le

temps

^de mortalite moyen

(TM^q) correspondant

^{aux deux}

types

^devirus

est

eomparable

^mais

depend

^{de la}

temperature:

^{les hautes}

temperatures

diminuent le

TMgn.

^{En conformite avec} les resultats obtenus au niveau de

l'infection du corps gras, l'effet d'une infection ä dose elevee^estde

prolonger

^le

TM^q comparativement

^äcelui obtenu lors d'une infection ä

dose

plus

^basse.