The influence of age on the pathogenesis of pox-virus diseases in mice and studies of the interferon response of mice to viruses

THE INFL1®lCE OF AGE ON '.mE PATHOGENESIS OF POXVIRUS DISEASES IN MICE AND STUDIES OF THE INTE~roN RESPONSE OF MICE 'It) VIRUSES

By

T. P. SUBRAHMANYAN

A thesis submitted for the degree of Doctor of Philosophy

in the

The results of the investigations described in this thesis are based upon work done by me without any help. The resul ts have been discussed i th Dr. C. A. Mime and many of the experiments prung from th se di oussions.

} P.

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lXJ1 J M f)f;--.l' <'1 'l

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~nderful is he who speaks of it.

Intelligent is he who learns it.

Blessed is he who, taught by a good teaoher, is able to understand it.

CONTENTS

Pag

A 1M 0 F TilE IlifVF.,,8 TI GA TI 0 N •••••••••••• ••• ,... 1

MATERIALS AND l~THODS

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N eone. tal thym.ectonty' ". ... 2

Splenectomy ••••••••••••••••••••••••••••••••••••••••••• 2 ColI otion of urine from mice

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Footpad mea urements ••••••••••••••••••••• _{• • •••••••} 3 Viruses

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Virus titrations

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Poxvirus neutralization tests • • • • • • • • • • • • • • • • • • • • • • $ • • 5 Poxvirus haemagglutination inhibition~~ antibody titrations

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Haemolysin t1trations .•••••••••••••••••••••••••••••••• 6 Gel diffu ion tests

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_{••••••••} 6

Fluoresoein conjugated antibody and immunofluorescent staining.. •••••••••••••••• ••••••••••••••••••••••••••• 6

Mouse emb~o fibroblast oultures •••••••••••••••••••••• 7 L cell cuI tures

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Chick embryo fibroblast oultures _{• • •}

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Peritoneal cell cultur s

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~ethod of preparation of materials for interferon assay • ••••••••••.••• • ... dI • .. • • • • • • • • • • • • • • • • • • • .. .. • 10

STUDIES ON 'IRE IN:FLUENCE OF .AGE ON THE PATHOGENESIS

OF POXVI

nus

DISEASES •••••••••••••••••••••••••••••••••••••• 12

General introduotion •••• o • • • • • • • • • • • • • • • • • • • • • • • • • • ~ • • 12

Introduction to the pr sent work •••••••••••••••••••••• 14

Susoeptibility of mice of d~fferent ages to poxviruses. 16

Lethal effects • • • • • • • • • • • • • • • • a 0 • • • • • • • • • • • • • • • • • 16

Infeotivity ••••••••••••••••••••••••••••••••••••••• 17

Pathogenesis ••••••••••••••••••• ~ ••••••••••••••••••••• 18 Growth of virus in young and adult mioe ••••••••••• 18 Immunofluoresoent and routine histologioal

studies ••••••••••••••••••••••••• a • • • • • • • _{• • •} • _{• • • •} • 20

Infeotion of liver by oiroulating virus ••••••••••• 22

HOst defence meohanisms _{• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •} 23 Immune response (a) Antibody respon e... 23

(b) Immune footsw lling and

resistance to ohallenge •••••• 24 Interfe on response ••••••• • • • • • • • • • • • • • a 0 • • • • • • • 24

Misoel1aneous faotors of possible importanoe in the

greater susceptibility of young mioe ••••••••••••••••• 25

Behaviour of peritoneal maorophages ••••••••••••••• 25

Ciroulating antigen ••••••••••••••••••••••••••• 27

Influence of intest1na1 organisms •••••••••••••••• 28

Attempts to depress the resista.noe of adul t mioe to

the lethal effeots of oowpox and eotre ' 1ia viruses $ . 30

Pag Effect of Thiotepa _••

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33 Effect of splenectomy •••••••••••••••••••••••••••• 34

Effect of cold environment ••

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35 Effeot of pregnancy .•••••••••••••••••••.•••••••••• 35

Disous ion ••••••

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35 Interferon production ••••••••••••••••••••••••••••• 36 Immune re ponse

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38 Viraemia

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42 Barriers

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Tissue susoeptibility •••••••••••••••••••••••••••• 45

Miscellaneous factors • • • • • • • • • • • • • • • • • • • • • • • • • • • a • 46

Neonatal thym otomy and other attempts to lower

the resistance of adult mioe to poxviruses ••• •••• 48

Cause of death •••••••••••••••••••••••••••••••• _{• ••} 49

S Ul1lDlary ••••• .... It .. • • • • • • • • • • • • .. • • • • • • • .. .. • • • • • • • • • .. II • • • • 50

Age-dependent resistano 50

Neonatal thymectomy •••••••••••.•••••••••••••••••• 5

STO'DIES 0 THE IN RFERON RESPONSE OF MIe", 10 VIRUS J~

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G n ral introduotion • • • a • .. .. .. • • • .. • • .. .. • • • • • • .. • • • • • • • • • • • 52 Produo ion

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53 Bl.osynthe is _{• • • • • • • • • • • • • • • • • • • • • • • • •} ~ • • • • • • • • • 0 55 Effect of interferon and other agents on

in rferon produotion •• ~ ••••••••••••••••••••• ~. _•• 57 Properties of interf ron _{• •}

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Interferon and cytopathog nicity of viruses ••••••• 64

Role of interferon in virus diseas s _{• • • • • • •} • • • • • • • 65 Introduotion to the pr sent exp riments ••••••••••••••• 67 Prelim1nary experiment •••••••••••••••••••••••••••••• 69

Comparison of the interferon response of

different strains of mice . a • • • • • • • • • • a . e • • •

Comparison of the plaque r duotion and CPE

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1nhibition methods for int rferon assay •••••••••• 70

Comparison of Sen[iki Forest and

enoephalomyo-carditis viruses •••••••••••••••••••••••••••••••••• 71

Compar1son of mouse embryo fibroblast and

L oel1s ••••••••••••••••••••••••••••••••••••••••••• 71

Attempt to 1norease the sens1tivity of the

plaque reduotion test in JlX)use embryo fibroblasts.. 71

R produo bility of the plaque reduction test •••••• 72 Therl'OO ta bili ty 0

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moue interf ron _{• • • • • • • • •• ••••} 72

Importanoe of removing particles of inducing virus

from interferon preparations •••••••••••••••••••••• 73

Clearanoe of intravenously injeoted influenza

virus ••• • • • • • • • • • • • • • • e • • • • • • • • • • • • • • • • • • • • • • • • • 74

Absence of interferon-like inhibitors in blood

and tissue .* •••••••••••••••••••••••••••••••• 74 Absence of effeot of normal blood and issues on

interferon activity •••••••••••••••••••••••••••••• 74

Fate of ntravenouslyadmini tered int rreron ••••••• 75

Di appearanc of int rferon from th circulation.. 75

Pres nee of injeoted interferon in th tissues ••• 75

Page

Distribution of interferon 1n virus-infeoted mic _{•••••} 78

'/e t Nile virus •• ~ ... " .... • _{• • • • • • • • • • •} • 79

Ectromelia virus (a) Footpad injection •••••••••••• 79

(b) Intravenous injection •••••••• 80

Interreron pr duction in ndce after the intravenous

injection of influenza virus ••••••••••••••••••••• ".,,~ 80 The height and duration of the interferon response. 81

The relationship between particle dose and

interferon re pon e •••••••••••••••••••••••• ~~.... 81 Interreron response fter

two

doses of virus _{• • • • • •}

of interferon as Production nd distributio

determined at 4~ hours •• • ••••••••••••••••••••••

lnt rferon olearance in mice tre ted with

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influenza virus •••••••••••••••••••• " ••••••••••••• 84

tudies on the possible sources of interferon pres nt

in mice injeoted with influenza virus •••• " •••••••••••• 85

Blood •••••••••••••••••••••••••••••••• "." ••••

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Sple n •••••••••••••••••••••••••••••••••••••••••••• 87

Liver ... .

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88 Histologioal study of the influenoe of interferon on

the cour e of ectromelia virus infeotion in mioe •••• ~. 90 Interferon produotion by peritoneal oells ••••••••••••• 93

Importanoe of the induoing virus ... 94

Induction of interferon production by Newc stle

disease viru •• ,,~ ••••• ~ ••• ~. • ••• ~ ••• _{• • • •} 98

Sensitivity of maorophe.ges to the aotl.on of

interferon ••••••••••••••••••••••••••••• • •••••••• 99

D1seU6sion •• _{• •••••••• • •••} "

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the mouse ••••••••••••••••••••••••••••••••••••••••• 102 Produotion 0 f 1n terferon in vi \TO • • • • • • • • • • • • • • • • • 104

Rapid interferon r spon e to intravenously

1njeoted viruse •••••••••••••••••••••••••••••••••• 106

Souroes of the rapid interferon

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108 lAacrophages •••••••••••••••••••••••••••••••••• 108 Liver •••••••••••••••••••••••••••••••• • • • • • • • • I I I Spleen •••••••••••••••••••••••••••••••••••••••• 112

Blood oe11s • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 113 Lllng •••••••••••••••••••••••••• ., ••••••••••••••• 114 Action of interferon 1n vi 0 against ectromelia

virus infection ••••••••••••••••••••••••••••••••••• 114 S~r~ •••••••••••••••••••••••• .,., ••••••••••••••••••••• 115

CONCLUDING REMARKS

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BIBLIOG~HY

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121

The course of a virus disease in an animal is determined by the

in r etion of the virus with many host factors which include the

specific l.nnnune response and a variety 0 f known and unknown

non-specific influences. In addition to the immune response, the age of

the host and interferon produotion by the host are likely to be

important. The present work describes a study of the influence of

age on poxvirus diseases, and experiments on the origin, fate, and

action of interferon.

The a~ of the investigation was thus twofold~ Firstly, it

sought to find out the reason for the greater susceptibili~ of

young mice to the lethal effects 0 f poxviruses. Secondly , it

attempted to determine the sites of interferon production in

virus-infected mice, the fate of interferon in the body and the effect of

interf ron on the oourse of ectromelia virus infection in mioe.

Since the investigation had two different though not mutually

exclusive objectives, this thesl.s is presented in two parts. The

first part deals with age-dep ndent resi tance of mice to the lethal

eff cts of poxviruses and the second with studies on the produotion,

Mice - Multicoloured, outbred mioe of th alter and ~iza H 11

In tl. tute train were u ed in m st experim nts Inbred mc 0

r

the

C

57Bl nd eBA trains were us d in some :xp riments. Mice of th

C

S7Bl and

eRA

trains wer small r than Hall Institute mice of the

arne age (T bl e 2).

Whil mice used in I ny experiment were reared under ordinary

(0) conditions without sp oia1 preoautions, mioe for some xperiments

wer rear d under low pathogen (LP) condition. The LP eolon~ of

utbred and nbred mice were start d with mioe del~ver d by Cae arean

eati n and nursed by rat or LP muse foster thers. The LP mice

were handled i.n special ro ms by a single p r on until used in the

xperiment •

In the experim nts on enterio baot ria (page 28) and on e

-na 1 thymectomy (pages 30 to 33 ), the mouse food oube contain d

0.09% chI r ~cetin and 0.05% achro~oin.

Neonatal t ymectoSf - This as done by M1.11er's

thod (1960).

Th flb enc the thymu we. verified at the end of the expert nt by utop y end maoro copic examine. ton.

Spl

n

etomw

-

Thi

W 8 don by ext rioriz~ng the spleen thr ugh

a retrop riton a1 incision nd r moving it fter ligatin~ th pediel •

Th bsenoe f auxi11ia~ p1eens 8 check d at the end f th

xp r nt by autop y and ero epic examina ti n.

Colleotion of urine f m mioe - This s don by kneadin~ the

3

Footpad me ere me sured by

the techniqu de crlbed by Aokerman (1964) with dial g uge c lipers

(Schn 11 taster, Sy tam Kroplin, 'IYPe A .02 T B.C. K "plin G.m. b.h.,

Sohluchtern, Res en, Gar ny), which read to 0 05 mm.

Viruse - (a) Ectromelia virus - The two strains of ectromeli

virus used have been d soribed by Fenner (1949b). The Hempste d

mous train deriving by mouse pas sag only from the 0 riginal viru-lent strain i olat d by ~rchel (1930) was passed onoe in egg and u ad s a chorioallantoic membrane preparation. The Hampste d egg

train is much Ie s virul nt than the Hampstead mou e strain from

whioh

it

was d rived by serial pas age in eggs. It had been pa sed

about 60 times when used by ~enner (1949b) and an unknown number of

times S1nce then. It was used as a chorioallantoic mbrane pre

ara-tion.

(b) Cowp x and v ccinia Tirus s - The Brighton s train of cowpox · ru

nd the V-MH end 7N strains of vaccinia virus (Fenner, 1958) w re

u ed as chorioallanwic membrane pr parations.

(0) Influenza virus - Fully nrectious allantoic fluid cont ining the

PR 8 strain of J.nfluenza viru s us ed in mo st expe riment s, but

ohicken red oell el tea of the BEL strain stor d at 20 _{er used in}

me experJ.ments. Th se eluates did not contain detectable amunts

of infeotious virus but h~d at Ie t 104 un1ts of baa gglutin n per (d) Sendai r - The Sendai t in of parainfluenza 1 virus obtain d

from Dr. D. O. White Ibourne, s used and stock virus W oontei ned

in allantoic fluid from infected eggs.

(e) N castle dis ase virus - A stra1n of Newoe. tle dise se -rus

obtained from Dr E. L Frenoh Melbourne, s used and stock vi us

s contain d in allantoic fluid from infected eggs.

(f) e t Nile virus - A suokling use brain preparation of the

Sara-fend train, p sed six times n mouse brains ince 1so1ation, as

u ad.

(g) Semlik1 Fore t virus - A strain of virus obtained from the

Rockefeller oundation Virus Laborator,y and passed 20 time in moue

brains and onoe in ohick embryo fibroblasts sinoe isolationg was used.

Stock virus w s contained in the m dium from infected chiok embryo

fibrobla ts. Infeoted adult mouse seMlln coll oted 24 hours aft r

intravenous inooulation of the iru was used in one experiment on

interferon produotion by .peritoneal 0 lis.

(h) Encephalolf.\V'Ccat'di tis virus- A strain of the virus obtained from

Dr. K. A. O. E1lem w s ueed. Stock virus was oontained in the dium

from inrected mouse embr,yo fibrobl ts.

Virus titrati ns - Poxvirusee ere titrated on the ohorio llantoic

membranes of ll-day old chick embryos by the teohnique of Watwood

tal. (1957) using 6 eggs per diluti n.

The

eggs were incubated at

36° and p cks oounted on the second day in the case of 0

cainia virus I and on the third day in the case f eatro Ii r ..

e t i l e virus was titrated on ohiok embryo fibrobl ate by th

5

Infectiou and noninfectious prepa tiona 0 f infl u nza viru

ere titrated for hae gglutinin by testing s rial 2-fold dilution

for bility to agglutinate ch1cken red oells. The ndpoint w the

highest dilut on at hieh partial haema glutination oeeurr d; th

reoiprooal of the ndpoint dilution was taken as th number of

ha gglutinating units (HAU) pre ent in 0.25 ~ (test volume) of th

undilu d material. The numb r f influ nza 'Virus partiel s pr ent

in a gi en dose was caloulated on the basis of a particle : HAU ratio

of 107 : 1 (Faz lea de St.Groth and Webster. 1963) ..

Poxvirus n utr lization tests - Sar w re tested fbr the pres noe

of n utralizing antibody using the V-MH strain of vacoinia virus as

antigen. S ra were diluted 1 in 5, except when otherwise tated and

ml.xed with equal volume of a virus dilution adjusted to contain

bout 100 pf'u of the virus; nOrln8.1 mous serum was used f r the virus

oontrol. E oh mixture s incubated at 370 _{for 90 minutes and then}

1nooulated 1nto 6 eggs. The neutral zing power of a serumw s expr sa d

s the per cent reduotion in oak hen compared ith the con Is.

Po xvi rUB ha magglutin tion inhibition (HAl) antibo~y titr, ti ns

-S ra from oowpox infected miee were heated at 56 for 30 minutes and

soreened f; r th pr 8 no of nonspeoific ha gglutinin8~ aoh arum

or the te t d luted in 2- 01 step in 0.25 ml vol Ulnes f

diluent (1% normal rabbit serum. in no mal ine) i ersp:x

hae gglutin ti n trays. To oh oup, 2 HAU 0 cowpox virus r the

for 20 minutes One drop (0.025 ml)

r

a 5% sp nsion of vacoini

-u oeptlble fowl red cells as added 'bJ e ch cup and after inoubation

for 20 mdnut s at 370s th t ys were haken and re1noubated for 40

minut s before readi g. 'I'he titr W' s express d as the eciprocal of

the highest diluti n of the s rum which partially inhibits ha magglutin

a-tl n.

Haemoly in titr t1 ns - Sera for the test re diluted in 2-fold

tep in 0.25 ml volumes of calcium-magnesium aline, mixed ith 4

uni ts of guinea pip; serum complement (0.025 ml) end a 2% suspension of

sheep red c 11s (0.05 ml).

The

trays ere shaken ell, inoubated at

370 and re d after EO minutes It !he titre was xpr s ad s the

oipro-oal of the highest dilution of the erum at which parti 1 haemolysis

oocurred

Gel diffusion test - The tests ere done on micros ope s11d s and

the diffusion medium was a mixture of equal parts of 3% agar, 3%

odium azide and 0.9% EDTA. Pr cipitin lin ere seen after 48 hours

at room tempe ature in a humid oh.alrh r. The gel lay rs w r Wi h d

and stained with Biebrich c rlet fbr better visualiz ti n.

Fluore 0 in conjugate~ antibody and immunoflu rescent at ining

-The globulin in a potent nti ocinia serum were precipitat d with

methanol, redia 0 ved and oonjugated i th fluore oein isothiocya te

(Syl'Vane. Chemical Company Orang New J rsey J U.S. ). Exe ss

fl uore 0 in was remQ-ved by di ly i against buffe, d salin (pH 7.2

at 40 _{The conjugate we. then ad orbed with rabbit I1ver nd c 10k}

7

nd u ed after the ddition of one-tenth velum of Rhodamin

oonju-gated bovin serum albumin (Microbiologioal Associates, Bethesda,

ryland, U.S.-A.).

Ti sues for immunofluoresoent examination were snap-frozen in

liquid-nitrogen-cooled contain rs, and sectioned on a cryostat at 6

f·

~e sections were dried under a fan, fixed l.n aoeton and stained.

Fluor soent ob ervations wer made with a Zeiss microscope

quipped for fluoresc nt mioroscopy, using an Osram HB 200 m rcury

vapour lamp as the ultraviolet sourc •

Mouse embryo fibroblast cuI tures - Secondary cuI tures were made

from 15- to 16-day nnuse embryo in 5 cm petri di hes by seeding

6

2 x 10 cells in 5 m1 of growth medium, or in screw-capped tub s by

eeding 4 x 105 cells in 1 ~ of medium. The gro th medium was

Eagle's minimum asential medium containing 7% calf serum (heated at

56° for 30 minutes). The monolayers w re confluent and ready for use by 48 to 72 hours.

Xhe

petri dish cultur s used in the plaque

a ays ere ov rlaid after inf ction wi th Earle's saline oontain·ng

0.5% laotalbumin hydrolysate, 0 1% Y ast xtract, 0.1% bovin pl sma

albumin fraotion V, 2% oalf s rum (heated at 56° for 30 minute),

0.0 d xtran and 0 75% agar.

L cell cuI ture - L oells of strain 929 obtain d from

Microbiologies. As oeiates, Bethesda, Maryland, UsS • • , wer cultured

5 om p tri dishes using the sam methods growth medium and overlay

Chiok embryo fibroblast oultures - Primary oultures of chiok

embryo fibroblasts ere grown from deoapitated II-day chiok emb~os n

5 om petri dishes. The growth medium as Hanks' ESS oontaining 0.8.%

laotalbumin hydrolysate and 8% oalf serum (heated at 560 _{for 30}

minute). The agar overlay was the same as that used for mouse

emb~o fibroblasts. but did not oontain serum.

Peritoneal oell cultures - (a) Maorophage cultures s Peritoneal

washings were obtained from untreated (unstimulated) mice by the

technique described by Mims (1964b). The medium oonsisted of Hanks'

BSS th 20% calf serum (heated at 5So for 30 minutes) and 0.5%

laotalb~n hydrolysate. In some experiments, the oells were cultured

in "ring' chambers oonsisting of pyrex gla s rings sealed to

mioro-soope slides ith petroleum jelly (Cairns. 1960); the rings received

2 x 105 oell in 0.2 ml of medium. In other experiments, 2 to

6 x 106 oells were oultured 1n petri d1shes oonta1ning 5 m[ of medium,

or in sorew-capped bottles oontaining 8 to 10 ml of medium. After

1ncubation for several hours at 37 , when the maorophages had adhered

to glass, cells not stioking to glass were washed off (Mackaness, 1962).

For infeotion, virus as u ually added to the full volume 0 f the

medium, adsorptio in a small volume being carried out only hen

tated. At least 2 replicates ere used in eaoh test.

In fluore oent antibody studie , "ring!f cuI tures were incubated

wi h virus for 8 to 20 hours. The medium was then remowd and the

9

the cells were fixed in acetone and stained with f.luoresce~n-conjugated

antibody. At Ie st 400 cells ere examined for the pre ence of antigen

in each 0 the duplioate slides.

In the experiment to determine the e..m.ount of infeotious virus

produced by maorophages, cultures in petri dishes were used.

They

were 1nfected by 1ncubating with 106•3 pfu of attenuated ectromelia

virus in 0.1 m1 of medl.urn for 3 hours at 37°. The cul t-ures er then

ashed thrice, refed with 5 m1 of fresh medium, and incubated for a

further 24 hours at 37°. The whole oul tures wer then stored froz n

at _600 until titrated.

In the experiments on interferon production by macrophages, petri

di h and bottle cultures were used. Details of virus used and method

of infection re given in the section of interfer0n (Tables 26, 27). The

cultures were freeze-thawed before prooessing for 1nterferon a say.

(b) Stimulated peritoneal washings: Peritoneal exudates were induced

by the intraperi ton a1 injection of 1 to 2 ml 0 f sterile nutrient

broth (Dirao Laboratories , Detroit, Mi~higan, U. S.A.). Peri to neal

washings er oolleot d 20 to 24 hours later; on one occasion, a

dirf r ntial count showed 66% large mononucl ar cells, 23% small

mononuclear cells, 9.8% polymorphonuclear cells and 0.4% eosinophil '.

The c lIs er oul tored in petri dishes or in screw-capped bottles and

infected by adding viru to the medium wi thin 30 minutes of collection.

After appropri te incubation the cultures were freeze-tha ed, exoept

Method of preparation of materials for interferon assay

-aterials for interferon assay ere dialysed at 40 against glycine-HOI buffer (pH 2 0) overnight and returned to pH 7.2 by further dialysis against everal ohanges of Dulbecoo's PBS at 4°. The dialysed material was centr1fuged at lOO,OOOg for at least 60 minutes in a Spinco model L preparative ultracentrifuge, and the supernatant was assayed for

1nterferon activity. In some exper1ments on the sera of

influenza-treated mice, dialysis against acid buffer was omitted, since these sera were unlikely to contain inhibitory amounts of virus (this thesis, page 74).

Preliminary exper1ments showed that treatments involved in preparing material for interferon assay did not affect interferon

titreso A 10% suspension of brains of mice infected 11th West Nile virus was divided into 2 portions.

One

portion was processed as usual, while the other one was only centrifuged at lOO,OOOg for 90 minutes

to remove the virus. Both.preparations gave identioal titr s of 800 units per ml. In another experiment, it me found that when stock

interferon was reprocessed, its titre remained unchanged.

Standard interferon - A preparation of interferon was made from the bra1ns of adult mice infected ~ntraoerebrally with about 600 LD50 of West Nile virus and killed on the fourth day when the brains are

known to oontain large amounts of interferon (Finter, 1964d). A 10% suspension of the 1nfect d brains was processed for interferon

assay as describ d in the previous section (acid treatment and

80

70

c

0

10

₆₀ E

L-0

'+-Q.) :::::s

0-co c. ₅₀

""'-0

c

0

...

.

-..0 ..c

40 c

()--.!2

30' ! -. I

3 4 5

log10 reciproca

1 d

i

1

ution

[image:23.829.25.815.18.1008.2]

~In~t~e~r~f~e~ro~n~~~~~~~ __ ~ - Interferon was assayed by th

fo1l ring stand rd procedur t

Confluent s condary oul tur s of mouse embry fibrobla t were

o

treated for 5 hours at 37 w1th 5 m[ of th appropriate dilution of

th D8 trial fb r a Bay in E gle' s medium. The fluid was then r moved

and the JOOnolayer inf eted with approxina t ly fl) pfu of Semllki

Forest virus in 0.1 ml of medium. After absorption for 75 minutes at

370 [I overlay medium was added. Two days later, the plates ere

stained with 1:20 000 neutral red for 2 to 3 hours at 37°, and plaques

counted. At lea t 5 plates wer used for each dilution and the average

pI que count oalculated in eaoh case. The percentage inhibition of

plaque counts in the treated plates, when oompar d to the average

plRque count in untreated control plates~ was oalculated for aoh

dilution. Results from a repres ntative test on standard interferon

illustrates the linear reI tionship observed between th p roentag

inhibi tion and the logarithm of he reciproca.l of the dilution over the

r ng been 30 and 80% inhibiti n (Fig_ 1). In each test a similar

graph s used to estimate the dilution at whioh th plaque count in

the treated plates would have been reduced to 50% of that in oontrol

plat • The reoiprooal of this dilution was d fined as the number of

un1ts of 1nterferon p sent in 5 ml of the starting naterial. Thus,

one unit of 1nterferon was defined as th amount contain d in 5 ~,

which reduoed the plaque count by 50%. For instanoe, if 5 ~ of a

1:100 dilution of an 1nterferon prepar tion reduced the plaque count

by 50%, the interferon titre of the preparation s taken to be 100

STUDIES ON AGE DEPENDENT RESISTANCE

GENERAL INTHDDUCTION

It has been recognized ibr

many

years that virus diseases a

gen rally more ev re in young than in adul t animal s. Andervont

(1927, 1929) reported that newly hatohed ohioks and 12-

to

14-day old

mioe were more susoeptible than adults

to

the lethal effects of

sub-outaneou ly inooulated neurovaccinia virus and herpes simplex virus

re pectiv lye Similar age-related effects have since been described

in the oase of several virus disease in a number of different speoies

of an~mal (Sigel, 1952). An important practioal applioation of

reater susoeptibility of young animals to virus dis as s is the u

of ohiok embryos and l.nfant

_..

mo in virus ark •

1 though young animals are gan rally more susceptible than adults

to virus disease, this is not invariably 0 (Sigel, 1952). For

instanc , the Lansing strain of poliomyelitis virus kills adul t mice

more re dily than young mice (Sabin, 1950; Da1ldorf, 1950); thi,

viru , however, is more lethal to baby cotton rat than to adu1 ts

(Pinto, 1948) 0 that the inver ion in age effect is not absolut •

dul t mioe are also more susoeptibl e than infanta to the 1 thal effects

of lymphocytic ohoriomeningi tis virus (1 i -mGY 1951) end thel.r gr ater

u 0 ptibility is thought

to

be due to their immune response being

uperior to that of infant mioe (Hotohin, 1962). Another 1nt resting

09. is that of the C nnecticut-5 train of Coxa one virus. Mioe

13

r i tant b een 5 nd 21 days and then moderat ly susceptibl unt11

8 or 9 we ks old (Pappenhei r e al., 1951); th newborn mic develop

Ie ions in y orgens, hil the old r mice in the s cond period of

leotive damag to the pancreas.

enhanoed usc ptibill.ty . how

The early de eriptive ork n g -dependent r sistance of animal s

to 'rlru di ea e soon foIl ed by ttempt to anal se the baS1C

elem nts constituting the U ge factor". Sabin and Olitsky (1937a~b;

1938 ,b,e) and S bin (1941) desar b d the development of barri rs to

the spr d of v iou1ar stom titis virus and equine encepha10my litis

virus through the body as th mice grew older. Subsequent work ith

a numb r f viru disee.ses ha made i t c1 ar that no single mechanism

aooount for e ry oa e of age-dependent re i ' tane to virus di a s~

For in tanc, hi1e the s r ad of v sieular tomatitis virus l.n adult

mic i limit d by barriers located at various plaoes in the body

(S bin and 01 tsky, 1937a,b; 1938a~b), re i tanoe of adult hamsters

and mice to mumps virus is thought to be due to their antibody respon

eing sup rior to that of susoeptible young animal (Over and

Kilham, 195 ; Ov r

an.

1954) and differences in re ist nee of mio of

diff rent ag s to Coxsaokie Bl virus have be n hown to be paralleled

by differ no

1n

interferon roduction (He1neberg at al., 1984).

Yet an ther po

who und that

ib1 meohani m ha been sugg at d by Johnson (1964)

rophages from resistant adult mice unlik tho from

usc tib1 young mice failed to spr ad herpes virus infection. That

y c rs ar involved in e-depend nt r istano to virus di e.. es

of re is nee doe not app ar at th same age for all viru dis ase •

Thi

view is alBo supported by the dditional obs rvation that the

ohang in u ceptibility to differ nt viru es t kee pI ce at dirf rent

rate , being gradual in oas like herpes simplex virus infeotion in

e (Johns n~ 1964) and rapid in other 11k yellow fever viru

infection in mice (Bugher, 1941). Therefore, several faotors acting

ingly or tog ther determine the ooourr nee of usoeptibility or

re i tano to virus diseases. Thee factor include th immune response

(Ov rman and Ki1ham 1953; Ove n, 1954), interferon produotion

(s ioki 196~J Helneberg at al., 1964), development of bar iers to

th pread of infeotion (Sabin and Olitsky, 1937a,b; 1938a,b,o;

Sabin, 1941; Johnson, 1964) and factor like body tempe tur (Enders

and Pe rson, 1941). The partioular combination op rating in any

tanc ould depend on the virus he host and the pa.ttern of their

interaotion

INTroDUCTIO TO 'ffiE PRESENT EXfERIMEN S

Study of ag -dependent resi t nee to virus di ease is likely to

be more profitable in the ease of virus disea es whose pathogenesis

i ell understood~ Sine the pathogenesis and histopathog n S1S of poxvirus inf otions in miee have b

(Fenner 1949a Roberts 1962, Mims

n p rtioularly

1964a), it a

ell studied

aonsid r d likely

th t useful ·nformation on the effect of age on virus diseases could

b obtained by a study of poxvirus infeotions in mice of diff rent

15

is known to ~ncre e th nore ing age. In an arly tudy 0 f e.g

-d P n-d n resi tance to poxviru s Green (1935) found th during a

rabbitpox pidemdo the great at mort lit.y occurr d in rabbits betwe n

the ~g s of 4 nd 9 weeks. Fenner (19490) found that 10- t l4-d Y

old ce w re killed more readily than adults by eotromelia virus. He

found the interval between virus in culation nd invasion of intern 1

organs to be horter in susceptible young than in resistant adult mdee,

and ooncluded that the young animals died due to the shorter time

ilable for antlb dy produotion and per ps les efficient antibody

r ponse. Oerskov nd Andersen (1938, quoted by Fenner, 19490) found

th t intrader.mally in oulated vaccini virus rapidly invaded internal

organs in 3-day old rabbits, but even small increases in age ere

coompanied by slower spread of the virus. Soh 11 (1960) r ported

th t adult mice of the C Bl strain resisted the leth 1 efr ots of

57

otromel ia irus better than adul t mie 0

r

three other strains lJ but

th only vidence of superior resi t,ance in newborn C Bl mice as a

57

1 nger survi 1 tim th n in newborn mioe of a susceptible strain~

Mar nnikova nd Kaptsova (19 ) have reported that in racerebrally

inoculated variola virus killed mioe only if they are less than 12

days old~ The virus grew to higher titres in the brains of au oeptible

young mioe th in the brain f re istant adult mice, Virus B also

fo d ~n th lungs and kidney of young but not adult mice~

The pr sent work reports on the effect of age on the BUSO ptibility

of mice to the lethal ffeot of oowpox and ectromelia viru es. To

ee{

Mortality in Hall Institute mice of' different ages due to cowpox a.nd

attenuated eotromelia viruses inooulated into the footpad

-

-Mortal~t,y_ in Daoe inoculated with

Age of Mioe

(weeks) Cowpox Virus

100 pta 10,000 pf\1

1 ND ND

2 2/6 1/7

3 0/1 0/7

4 0/6 0/6

6 0/6 0/6

ND - Not don

pfu - Pock forming units

Numerator - number of deaths

Denominator - number of moe used.

-~-.

--Eotromelia Virus

10 ptu 100 pm

S/S 7/7

6/6 6/6

1/7 0/6

0/5 0/5

[image:30.840.20.825.18.990.2]

16

oomparative study was made of the pathogenesis of the disease in young

and adult mice. Factors like the immune response, interferon produotion,

the ease of infection of maorophages and their abil1ty to spread

in-fection were studied in the two groups. Young mice were treated with

broad speotrum antibiotics to alter their intestinal flora and the

effeot of the trea~~nt on their susceptibility to poxviruses as

studied. Experiments were done to determl.ne whether large amounts of

viral nntigen were present in the oirculation of young mioe only.

Attempts were made to depress the resistanoe of adult mioe

to

cowpox

virus by immunodepressive treatments including neonatal thymeotomy,

administration of the cytotoxio drug Thiotep and splenecto~.

Normal adult mice exposed to oold, and pregnant mioe were infected

l.th cowpox v~rus and observed to determine whether the resistanoe of

adult mioe was affected by oold or by pregnancy.

SUSCEPTIBILITY OF MICE OF DIFFERENT GES TO POXVIRUS S

Lethal effects - Hall Institute mioe of different ages ere

inoculated into the footpad with different doses of either cowpox

virus or the attenuated (Hampstead egg) strain of ectromelia virus.

The

mortali~r figures (Table 1) showed that these mice attained adu!t

levels of resistance to both viruses between 2 and 3 eeks of age.

Co ox virus was less virulent than the attenuated strain of e tromelia

virus. dult mice

cowpox virus even

re never killed by subcutaneously inoculated

TABLE 2

ge~dependent resistance to subcutaneous (footpad) inoculation of oowpox virus in 3 different strains of mice.

sa: ....

.

_ _ :u:-. _ :ue

.

Age of _{Mortal! ty in mice}

mice in Hall Institute _C

57Blaok CBA

weeks

Mean

103 • 4

*

4.4* 5.4* Mean 103 •4• _{104 • 4}

_*

_{105• 4}

_*

Mean _{103 • 4• 104 •4}

_*

_{105 • 4•}

weight 10 10 weight _weight

(gm) _{(gm) (gm)}

2 11 6/8 5/8 7/9 8 6/6 7/7 5/5 8 3/6 7/7 9/9

:3 11.5 0/5 0/5 0/5 15 0/5 3/5 4/5 14.2 0/3 0/4 0/1

4 22.5 0/5 0/5 0/5 20.3 0/5 0/5 0/5 18.5 0/5 0/5 0/2

-

-

-

,. ...

-r of pook fbr.mlng units inooulated. Numora tor - number of deaths.

[image:32.1050.18.1045.29.759.2]

TABLE 3

eet

Suseeptibili ty of 2- and 6-week old mioe to cowpox virus inooulated

by different routes

Mioe lo~ number

or

pf'u* required to kill 50% of the mice ••

subcutaneous intracerebral intranasal int ravenous

Young 2.6 1.4 2.3 ~4.9

Adult >7.3 308 6.9 ')' 5. 9

--- - "--

-• Pook forming units

[image:33.847.25.835.19.1007.2]

In nother exp riment, 10-fold di1uti ns of cowpox virus ere

inoculated into the footpads of Hall Institute, C 7B1 and eBA m10e of

differ nt e.g d mortality rates d termined. Th. r suIts (Ti bl 2)

showed that H 11 Institut nd eBA mie 8.equired adult levels of re-is tanoe b tween 2 and 3 weeks 0 f ge, but C BI mioe did no t ttair:

57

adult level of r si tance until a aek lat r~ I all subsequent

xperim nt , 2-we k old Hall Insti tate mie (young mioeU) were oompared

wi h 6-week old mie ("adult mieen) of the same strain. Th experi nts

on g - 1 ted resi tance to poxvirus were don with cowpox viru ~

The rfect of drrd.ni tering cowpox virus by various route as

then xamined. S ri 1 dilutions of the virus were inoculated into

g up of young and adult Hall Institute mice using 6 mioe per group,

and the number of pock forming units required to kl.ll 50% of the mice

det rmined. Th r suIts (Table 3) how d that young mic re more

su

c ptibl what ver th route of inf otien.

Infeot).

vi:t!Y -

The suboutan ou infecti vi ty 0 f oowpox virus in

young and adult mice s compared by inoculating lO-fold diluti ns of

th viru into th ir footpa.ds. Primary fb tpad swellJ.ng was scor d

as clinioal evid noe of infection (Fenner, 1948) nd on the fourteenth

day urv1vors w re ch 11enged into the oppo ite foot with 106•7 pfu

of virul nt eotrom lia virus (Hrunp tead mouse strain)" Ocourr no of

immune eiling in th challenged foot (Fenner, 1948) was m asured at

9 hours and the mic observed for a furth r 21 day, for d ath du to

~

I

I

TABLE 4

Production of primary footpad swelling (infectivity), mortality, and of immunity (immune footpad

swelling and resistance to virulent eotromelia virus challenge) in 2- and 6-week old mice

followint; subcutaneous infection with cowpox virus

eClIJ.on

Pri inf'

r--

Cbal1en~e

of survivors (106•7 pfu of virulent

rua)

loglO pfu·

I

Primary f'ootswellint; Mortality

Immune

footswelling** Eesistance to challenge

(survival)

Young Adult

Young

Adult Youn~ Adult Young Adult

1.6

5/8

2/5 1/8 0/5 0/7 2/5

4/7

2/6

2.6 8/8

4/5

_4/8

0/5 0/4 2/4

4/4

2/5

3.6 6/6 5/5 6/6 0/5 _••• 0/5

_•

_•• 1/5

4.6 5/5 5/5 4/5 0/5 1/1 2/5 1/1 4/5

5.6 _{2/2 5/5 2/2 0/5 ••• 3/5 • •• 3/5}

"

-* Pock forming units

Measured 9 hours after challenge.

Numerator ... Number of reactors in the columns on footswell1ng, number of deaths in the colwms on

mortality and the number of survivors in the oolumns on survival.

Denomina.tor .,. Total number of mice used •

[image:35.1047.135.1037.20.820.2]

7.3 (

nonl thaI to adult mice ev n when 10 pm were inoculated this thesis,

page 16 ) I but only 400 pfu killed half the young mice (T: ble 4). Both

groups er equally usceptible to infection as indicated by the

occurrence of primary footpad swelling (Table 4). None of the young

mice whioh surviv d 40 or 400 pfu of cowpox virus developed immune

footpad swelling when challenged but ome of the dults in comparabl

groups did; re istanee to the lethal effeots of the ohallenge was~

however, een in both the groups of mice (Table 4), even in those

surviving the smallest doses of oowpox virus.

Thus, as judged by the primary footpad swelling adult mioe are

a easily inf oted as young mioe by cowpox virus J but adults are mor

resi tant -00 the lethal effeots 0 f the virus. Survi ving young mice,

ho ever, fail d to d velop the immune footpad 5Welling r spon e although

they were able to resist challenge virus as re dily as adults.

PATHOGENESIS

In order to find a possible reason for the lethality of cowpox

virus in young but not in adult mio , studies wer made of virus

gro1th and histopathogenesis in young and adult mioe infect d with

oowpox virus II

Gro th 0 f virus :Ln young and adul t mi 0 - Cowpox virus w s

:Lnocula ted l.n to the footpads of young and adul t nuce, and footp d

sw lling, and virus titres in various tissues ere d te~ned daily.

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Fi~. 2. Results from 3 separate experiments showing the

development of footpad swelling and the growth of virus

in the inoculated footpad, liver and spleen, in young

and adul t mico inocula ted subcutaneous 1y wi th cowpox rus (virus t1ttes are expressed as the logarithm of

presented in Fig 2. Virus gro th was more rapid in the feet of

young mioe and was acoompani d by a gr ater d gree of footpad w ling.

P ak virus titr s in the feet of young mic ere higher than in adult

feet and after 4 d Y the titre fell in adult feet but not in the

t of young mioe. The liver and spleens of young mioe oontained

ubstantial amounts of virus J but very little was pres nt in these

organ in adult mice. In young mioe the infection also involved the

kin 0 f the general body surfaoe; a large area 0 f the skin taken from

the baok and abdom n of a siok young mouse oontained 106 pfu per gram,

and fooi of infeet10n in the skin were readily detected by

immuno-fluoresoent stain1ng in young mice but not in adults. In another

imilar experiment hole blood from young mioe was found to have a

virus titre of 104 pf'u per ml on the fourth day of infeotion and lesser

amounts on the sixth and ighth days· no vi rus as found in the blood

of adult mice at oorresponding times.

en 00 pox virus s inJected intravenously and blood virus

t tr deternun d a. t interval s, i t s found the. t virus was cleared

qually well by young and adult mice. Follow1ng an inJection of

106 pfu, 99% 0 f the injected virus was clear d wi thin 5 minutes by

young mce. This rate i oomparable to the olearanoe te of

pox-virus in adult mioe ( . , 1959a.). Therefore, th observed dir~ renoe

in vi r emie. b and adult mio was not due to differeno in

their ability to 01 r V1ru from th ciroulation.

20

sub tanti 11y gre t r virus multiplication at the inoculation 6ite~

virae~ and s~gnifioant invasion of the internal organs~ The virus

titres in th liv r and spleen h ever, were muoh 1 ss than tho e

found by enner (1948) in the organs of adult mice dying of aoute lOOusepox.

Immunofluoresoent and routine histological studie - Experim nts

were carried out to determin whether the pattern of infeotion in

you g mice s differ nt from that in adults at th histologioal

level. Groups of young and adult mic were inooulated into the

foot-pa i h 105•9 pfu of cowpox virus and killed on the sixth day hen

the young mio er iok Eaoh animal was utopsied and speci ns for immunofluo so nt studi s collected. At utopsy, adult mioe

appeared no 1 exoept for footpad sw lling and enlargement of 100901

lymph nodes. The young mice had, in addition to the above changes,

numerous d~screte fooi of necrosis in the liver; the bone marro ,

01 arly vi ible in the tail and sternum, was a deeper red than in

normal mice of the same age. There were no skin lesions, except in

th inoculat d feet.

Immunofluore oent examination of seotions of liver, sp1een9

oardiac mu' 01 , brain lung and of blood smears from adults did not

reve 1 the pr sene f viru antige in a. 0 f th m. Th sick young

oe on the other hand, showed l~ted involv' ment of certain orga s.

In th liver th r were large, discr te fooi of rluore c nt hepatic

cen reI veins. Even so 1iv r involvement was limited nd fooa1, and

1 than a quarter of the hepatio cells were inf cted. lnr ct d

c lIs ere not seen in adult livers; and even after the inj otlon of

large dose (107 pfu or more) by the intravenou route, when scattered

fooi were seen in adult liv rs the number of foci seen was 1 ss than

in the liver of subcutaneously inf cted young mioe. There was almost

oonflu nt fluoresc nce on the serosal surface of the spleen of young

moe, involving the capsul and extending down the trabeculae. Within

th spleen, infection was oonfined to the red pulp. The skin of the

b ok showed discrete foci of infected oells involving the dermis and

the pide~s. Infeot d oells were also pres nt in the kidn ys and

in bone marro smear but rarely if ever, in the lung, brain, oardiac

musole thymu I lymph nodes, intestinal wall or in blood mears.

Bbutine hi tologioal sect~ons from formalin-fixedj

paraffin-mb dded tissues 0

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sick young mice and normal mioe 0 f the s me age

w re stained ith haematoxylin and eosin and examined. In the

liver of s10k young mice th re were neorotio foci corresponding to

the infec ad area en after immunofluor c nt taining. The spleens

re enlarged and sho ad gross depletion of oells, partioularly in

the follicle. Th re as necrosis in the oapsule and trabeoulae

oorrespond1ng as in the liv r, to th areas stained by fluorescent

antibody. There re hardly any morphologioally intaot lymphoid

follioles Pykno i nd karyorhexis were oommon in megakaryooytese

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TABLE 5

Infeotion ot the livers of 2- and 6-week old mice with a large dose

of intravenously inocula ted cowpox virus

Mean number of infected _{Number of clusters of}

Age of Mice _{cells per standard field infeoted cells per}

(weeks) _{standa.rd field}

-10 hrs. 24 hrs. 56 hrs. 10 brs. _{24 hrs. 56 bra.}

2 42 _{70* 756-tW* 0 10 21}

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6 _{1 2 364 0}

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One of the 3 livers was more heavily infected than the others and contained 210 infected oells per field.

**

Infection in one out of 3 livers was semi-confluent. It was not included in the oalculations and henoe the fi~res underestimate