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CVO STUDIES U4TU NEWCASTLE DISEASE VIRUS

by

DENNIS JOHNQALEXANDER

Department of Virology

Royal Postgraduate Medical School

London.

Ph.D. Thesis

University of London

1971

ABSTRACT

The object of this work was to investigate the cause of the

differences of virulence between Newcastle disease virus (NDV) strains,

with particular reference to the role of virus proteins.

Methods for the purification of NAV using zonal rotors are

described.

Separation of purified virus by acrylamide gel discelectrophoresis

after sodium dodecylsulphate (SDS) treatment revealed four structural

polypeptides which were similar in migration and proportion for all

strains. The molecular weights of the three major polypeptides were:

88,000, 58,000 and 42,000; haemagglutinin, ribonucleoprotein and

neuraminidase were associated respectively with these polypeptides.

The minor polypeptide, molecular weight) 200,000, was not associated

with any known component or property of the virus.

The four structural proteins and tWo other 'non-structural'

proteins were detectable in infected chick cells. Strain Herts

induced two other non-structural proteins but later than 10HR,after

infection. Virus induced proteins were detectable earliest after

infection with virulent strains.

Neither the biochethical properties of neuraminidase nor the

amount per virion showed any relationship to virulence. The cell-

associated titre of neuraminidase in chorioallantoic membranes 22 HR

after infection was directly related to the virulence of the infecting

strain.

The extent of cell fusion and haemadaorption, and inhibition of

cellular protein synthesis in infected chick embryo cells were related

to the virulence of the infecting strain. All three required

protein synthesis in the first four hours of infection.

Congo red gave some protection of chick embryos against low

but lethal, doses of virulent strains. Very high doses of avirulent

virus showed greatly increased lethality to chick embryos in the

presence of congo red.

The cumulative production of RNA in infected cells treated

with actinomycin D was directly related to the virulence of the

infecting strain.

It is concluded that the virulence of NDV strains is a result

of the rate of production of one or more virus product in infected

cells.

ACKNOWLEDGEMENTS

This work was aided by grants from the Agricultural Research

Council and Action for the Crippled Child (Polio Research Fund).

I am indebted to Dr. Peter Reeve for his constant help, encouragement

and guidance throughout the course of this work and the preparation of

this thesis.

I would also like to thank: Professor A.P. Waterson for his

advice and encouragement; Mr. W.H. Allan for supplying the virus

strains used in this work and his helpful advice and discussions;

Mr. Peter Lister for his help and advice particularly during the

preparation of the figures; Miss Gill Pope and Miss Joy Pacey for

their help, and all those who during the course of this work have

offered advice and criticism.

CONTENTS

PAGE

I REVIEW AND INTRODUCTION

A. History 1

B. Morphology structure and chemistry 3

1. The virion 3

2. The substructures 3

a) Ribonucleoprotein 3

b) The envelope 5

C. Classification of NDV - a myxovirus 8

D. Replication of NDV 12

1. Adsorption 12

2. Penetration 12

3. Eclipse phase 13

a) General events 13

b) Types of NDV-specified RNA produced during infection 13

c) Role of complementary RNA 14

d) NDV-specified RNA-dependent RNA polymerise 15

4. Virus maturation and release 16

E. Virulence - range, and methods of assessment 18

P. Strain differences and virulence 21

Virulence definition 21

Foreword 22

1. Morphology and virulence 23

2. Serology 23

3. Biophysical properties 24

4. Cytopathogenecity 24

a) Haemadsorption 25

b) Syncytial formation - cell fusion from within 25

c) Plaque formation 26

d) Plaque size 26

e) Lysosome damage 28

f) Syncytial formation - cell fusion from without 28

3. Biological activities 30

a) Haemagglutinin 30

b) Neuraminidase 31

6. Virus multiplication 32

a) Growth rates 32

b) Growth site 35

7. Effects on cell metabolism 36

a) Protein and RNA synthesis 37

b) DNA synthesis 38

c) Lipid synthesis 39

8. Interferon and virulence 39

a) Interferon sensitivity 39

b) Interferon production 39

G. Effect of congo red on NDV infection 42

H. Virus specified proteins in NDV-infected cells 43

J. Purification of NDV 45

K. Objects of investigation 47

II MATERIALS AND METHODS 48

A. Reagents and equipment 48

1. Reagents 48

2. Equipment 50

B. Virus strains and virus techniques 52

1. Strains 52

2. Growth of virus 53

C. Cell culture techniques 54

1. Preparation of chick embryo fibroblast cells 54

2. Infection of cell monolayers 54

3. Staining cell monolayers 55

a) Giemsa - May GrUnwald 55

b) Acridine orange 55

D. Biological assays 57

1. Haemagglutinin 57

2. Infectivity 57

3. Plaque assay 57

4. Neuraminidase assay 57

5. Protein assay 58

6. Lactate dehydrogenase assay 58

7. Estimation of haemadsorption 58

8. Estimation of cell fusion 59

a) Size of syncytia 59

b) 7 polykaryocytosis 59

9. Estimation of cell-associated neuraminidase activity 59

E. Virus disruption 60

1. Sodium dodecyl sulphate-2-mercaptoethanol 60

2. Wean 20 at pH 10 60

3. Sodium demqcholate 61

F. Radioactive isotope counting techniques 62

1. Scintillation counter 62

2. Scintillation fluids 62

3. Estimation of uptake of radioactive isotope into coverelip cell. cultures 62

4. Estimation of radioactivity in gel slices 63

5._ Quench curve 63

6. Radioactive virus 65

G. Polyacrylamide gel discelectrophoresis 66

1. Preparation of 7.57. gels 66

2. Running 67

3. Staining 67

4. Estimation of polypeptide molecular weights 68

H. Double labelling techniques 69

J. Density gradient centrifugation - virus purification 71

A. Virus

III RESULTS

72 purification and biophysical properties

1. Linear sucrose gradient 72

2. Linear tartrate gradient 72

3. Association of viral properties to peaks 72

4. Discontinuous gradients 76

5. Recentrifugation of HA peaks 76

6. Nature of major and minor HA peaks 80

7. Separation under varying conditions 80

8. Estimation of virus purity 80

9. Behaviour of different strains of NDV on density gradients 84

10. Discussion of results 87

B. The structural proteins of NDV 89

1. Polyacrylamide gel discelectrophoresis after disruption by SDS 89

2. Polypeptide patterns of different strains 89

3. Radioactive labelled virus 89

4. Relative proportions of the polypeptides 95

5. Estimation of the molecular weight of the polypeptides 97

6. Identification of NDV polypeptides 97

a) Nucleocapsid polypeptide 97

b) Neuraminidase and haemagglutinin 100

C. Neuraminidase studies 108

1. Relationship of neuraminidase to HA and infectivity 108

2. Kinetic studies of virus-bound neuraminidase

108

3. Heat stability of virus-bound neuraminidase

113

4. Effect of Tween 20 at pH 10

113

5. Neuraminidase activity in infected chorioallantoic membranes

118

D. Cell fusion (from within) and haemadsorption

120

I. Haemadsorption

120

2. Cell fusion (from within)

127

3. Effect of inhibition of protein synthesis on virus induced cell fusion and haemadsorption

133

4. Effect

1. Effect

2. Effect

3. Effect

F. Effect of

E. Inhibition

of time of addition of FPA on cell fusion and haemadsorption

of cellular protein synthesis

of infection with different strains on cellular protein synthesis

of p-fluorophenylalanine on virus induced inhibition of cell protein synthesis

of time of addition of FPA on virus induced inhibition of cell protein synthesis

congo red on the mortality of eggs infected with NDV

133

137

137

139

139

142

G. The synthesis of NDV induced proteins 148

H. RNA production of infected cells in the presence of actinomycin D 157

IV DISCUSSION 159

A. Structural proteins 159

B. Neuraminidase studies 162

C. Cell fusion and haemadsorption 164

1. Haemadsorption 164

2. Cell fusion 164

3, Effect of inhibition of protein synthesis on cell fusion and_haemadsorption 166

D. Inhibition of cellular protein synthesis 167

E. Effect of congo red on the mortality of eggs infected with NDV 168

F. The synthesis of NDV induced proteins 169

G. Production of NDV specified RNA 172

H. General discussion 173

V CONCLUSIONS 176

VI SUGGESTIONS FOR NEITHER WORK 176

VII REFERENCES 178

I REVIEW AND INTRODUCTION

A. HISTORY

Newcastle disease was first recorded on the island of Java in

1926 (Kranevald, 1926) but first recognised as a virus infection

after a later -outbreak on a farm near Newcastle-on-Tyne (Doyle,

1927). These early outbreaks showed incubation periods from 4-11

days; respiratory and nervous signs (twitching and paralysis) as

well as general malaise were the moat apparent symptoms and mortality

rates were as high as 98-100%. Because of the high mortality the

early outbreaks in England were self-limiting. However in 1933 and

again in 1947 there were outbreaks of equal virulence. A milder

form of the disease also appeared in 1947 and has been endemic in

England since that time.

In 1944 it was recognised that a disease of chickens that had

prevailed in California for almost nine years called "chicken flu"

or "pneumoencephalitis" (Beach, 1943) was, in fact, Newcastle disease

(Beach, 1944). The delay in recognition was due to the entirely

different clinical features of the disease to those usually presented

by Newcastle disease. The disease was far milder than any form of

Newcastle disease known at that time, the respiratory and neurologi-

cal symptoms were notably mild and the mortality rate was often less

than 15%.

Newcastle disease is now known to be present in virtually every

country in the world (Levine, 1964) although in some countries, 2141.

Ireland and Australia the strains of virus present are particularly

avirulent. Bankowaki (1961) and McFerran (1966, unpublished) have

1

isolated virus strains of this kind which are symptomless in adult

birds, infection prior to isolation is detectable only by haema -

gglutination inhibition tests on the fowls serum.

In England since the early outbreaks the environment of the

chicken has greatly changed. Intensive rearing methods used in the

'broiler house' system haimeant that large flocks of many tens of

thousands of birds are kept. These flocks are indoors with the

birds in very close proximity to each other which gives ideal con-

ditions for infection to spread amongst a flock. It has been the

policy in EnglanU. since 1965 to control the endemic disease by

vaccination with inactivated virus; until Chi* time a policy of

slaughter, which is still maintained in Scotland. had been practiced.

Although several outbreaks have occurred since the introduction of

vaccination these were never extensive epidemics and the disease

VAS generally considered, perhaps too complacently, Co be under con-

trol.

In the autumn of 1970 a new outbreak of Newcastle disease

occurred in Essex which proceeded to spread throughout England as

an epidemic of unprecedented proportions, by February 1971 over

12,000,000 birds bad died. The virus unlike other strains of high

virulence is pneumotropl(ic rather than neurotropgic and the chick-

ens appear to die of respiratory malfunction (Allan, personal

communication). Conventional vaccination has had little success

in preventing the spread of the disease and in January 1971 the

Ministry of Agriculture gave permission for vaccination with live

virus in the form of the vaccinal strain B1. The effectiveness

of vaccination with this virus in controlling the present outbreak

has yet to be seen.

2

B. MORPHOLOGY STRUCTURE AND CHEMISTRY

1) The Virion

Viewed in the electron microscope using negative staining

techniquesI NDV populations can be seen to be grossly pleomorphic

(Schafer, Schramm and Traub, 1949), while individual virions have

no rigid structure and frequently appear disrupted on the grid.

The upper limits of the greatest dimensions of the NDV virion are

120-130 nm. (Waterson and Cruickshank, 1963)

Chemical analysis of pure NOV virions has shown the following

composition by weight: 677. protein, 1% RNA, 24% lipid, 77. carbo-

hydrate (Cunha et al, 1947). The viral RNA is surrounded by

protein to form a helical nucleocapsid (Kingsbury and Darlington,

1968). This nucleocapsid is itself contained inside a lipoprotein

membrane of host origin (Cruickshank, 1964) from which project

numerous regular spikes (Horne et al, 1960). The neuraminidase and

haemagglutinin activities are also associated with the envelope.

2) The substructures

a) Ribonucleoprotein (RNP)

Treatment of the virus with ether was shown to release the inner

component, the RNP (Schafer and Rott, 1959). The RNP of NDV is 17-

18 nm. wide has a central bole 4-5 nm. in diameter and a periodi-

city of 5 am. along the axis of the helix (Horne and Waterson, 1960).

Release of the RNP by ether tends to produce pieces of variable

length but osmotic lysis of NDV infected rhesus monkey kidney

cells gave a value of 1.06p for the intact length (Compans and

Choppin, 1967) and from deoxycholate treated virions 1.3-1.4p

(Kingsbury and Darlington, 1968).

3

RNP contains 9.3% RNA (Rott, Waterson and Reda, 1963) which

can be freed of the protein by treatment with sodium dodecylsul-

phate solution (Kingsbury and Darlington, 1968). Estimates of the

M.W. of the RNA from NDV vary from 5.8 x 106 (Nakajima and Obara,

1967) to 7.5 x 106 (Duesberg and Robinson, 1965). The sedimenta-

tion coefficient of the RNA in 0.05M Nadi, 0.01M sodium acetate

0.5% SDS was 49.2S increasing to 57S in 0.1M Nadi 0.01M Tris/HC1

and 0.001M EDTA (Adams, 1965, Duesberg and Robinson, 1965, Kingsbury,

1966). A low molecular weight component (36-4S) isolated was

assumed to be a degradation product of the 57S RNA. The isolated

RNA was easily degraded by RNase, suggesting it is entirely single

stranded RNA (Nakajima and Obara, 1967). Newcastle disease virus

has therefore a genome which is several times larger than that of

any other single strand RNA virus (except Sendai) which implies afar

greater genetic information content.

High molecular weight RNA isolates from the Beaudette C

strain of NDV examined by Kingsbury (1966) failed to show any sign

of infectivity in chick embryo monolayers, although isolated

Western equine encephalomyelitis virus RNA used as a control under

the same conditions did. However, infectivity was assessed by

ability to form plaques which in the NDV system is not necessarily

indicative of infection (Table 1).

The protein of the RNP has recently been shown by polyacryl-

amide gel electrophoresis techniques in the presence of SDS to con-

sist of a single polypeptide subunit (Evans and Kingsbury, 1969,

'bishop Cheyne and White, 1969 and Bikel and Duesberg, 1969).

Evans and Kingsbury (1969) and Bikel and Duesberg (1969) report

the molecular weight of the polypeptide sub unit as 62,000 daltons

while Haslam et al using a similar procedure quote 54,000 daltons

and estimate that the amount of thlsprotein per virion is 45% of

the total virion protein. All!these estimations of molecular

weight used the technique of Shapiro, vinuela and Maizel (1967),

and should be regarded only as approximations.

b) The envelope

Difficulties have arisen in recent years over the nomencla-

ture of myxovirus sub units. Originally haemagglutinin referred

to the rosette-like structures produced by ether treatment which

retained both haemagglutinating activity and neuraminidase activity

(Waterson, 1964). In the last few years subunits showing haema-

gglutinin activity have bean separated from those with neuramini-

dase activity (at least with influenza virus). The subunit possess-

ing haemagglutinin activity has been called haemagglutinin as well.

In this review haemagglutinin refers to the subunit protein which

possesses haemagglutinating activity and nothing else.

The envelope consists of protein, lipid and carbohydrate.

It is apparently derived from host cell membranes which have been

modified by the incorporation of virus specified material.

(Cruickshank, 1964, Hoyle, 1954). The envelope has an outer cover-

ing of spike-like projections 8O long, 10-158 wide and spaced 10-

1008 apart over the envelope surface. The spikes in this and

other mymoviruses are associated with the haemagglutinin activity

of the virion (Waterson, 1964, Biddle, 1968).

Because of the ease with which influenza can be degraded with

biologically active subunits, compared with NDV, moat studies of

myxovirus substructure have been with that virus (Laver, 1963, 1964,

Laver and Kilbourne, 1966, Laver and Valentine, 1969, Drzenfek,

5

Frank and Rott, 1968) and little has been done to isolate pure

biologically active subunits from NDV. Drzeniek and Rott (1963)

however treated purified, freeze-dried virus with n-butanol and

trypsin and extracted a non-haemagglutinating neuraminidase-con-

taining component which had a molecular weight of 200,000. It

was, luggested that this was a single molecule of neuraminidase,

however the component still induced neutralizing and haemagglutin-

ation inhibition antibody when given intravenously to rabbits

(Rott, 1964).

A study has been made of the biochemical properties of the

neuraminidases of two strains of NDV, Italian and Beaudette C and

it has been shown that although they are alike in their biochemical

properties, (Rott, Drzeniek, Saber and Reichert, 1966, Drzeniek,

Seto and Rott, 1966) NDV neuraminidases show significantly

different characteristics from most influenza neuraminidases.

Drzeniek (1967) has also shown that NDV neuraminidase is unlike

Vibrio cholerae neuraminidase, in particular NDV cleaves only 2-3'

linked sialolactose whereas Vibrio cholerae neuraminidase cleaves

both 2-3' and 2-6' linkages.

The actual relationships between haemagglutinin and neuramini-

dase and their structural positions on the envelope are not clear.

There is strong evidence•in the influenza system that haemagglutinin

and neuraminidase are proteins of separate entity (Drzeniek, Frank

and Rott, 1968, Laver and Valentine, 1969). However much of the

evidence comes from experiments employing recombinants of strains

which either have no neuraminidase activity or no haemagglutinin

activity. These do not necessarily lack the other protein but may

possess only an inactive form. The methods of disrupting the virion

6

to bring about separation of the biological activities are

usually very harsh (SDS treatment or_proteolytic enzyme action

in conjunction with a solvent) and could merely be separating

polypeptides of a single protein. Rott (1964) has suggested that

NDV neuraminidase may be a monovalent part of a polyvalent haema-

gglutinin.

Disc electrophoresis of NDV on acrylamide gels after SDS

treatment has revealed three major protein bands and several onts

smaller/(Evans and Kingsbury, 1969, Haslam, Cheyne and White,

1969, Bikel and Duesberg, 1969). Of the three major bands the

middle one is without doubt the RNP polypeptide while the other

two bands remain to be satisfactorily identified. The slowest

moving band M.W. 80,-90,000 has been associated with haemagglutinin

activity while the fastest moving band M.W. 38,-40,000 with neura-

minidase (Haslai et al, 1969). However in neither case was the

protein isolated and purified which are the ideal conditions for

this type of identification.

Several other biological activities associated with the OrAti

envelopey/haemolytic activity, ATPase and ADPase have been found

in preparatioo3 of NDV, these are now considered to be of host-

cell origin. (Neurath and Sokol, 1963, Neurath, 1965, Allison,

1967).

7

C. CLASSIFICATION OF NDV - A NWOVIRUS

Andreves, Bang and Burner (1955) suggested the name myxovirus

to include influenza. A, B, C, fowl plague, Newcastle disease

and mumps virus. All these viruses had distinct interactions with

mucoproteins (haemagglutination, and elution due to neuraminidase)

and the prefix myxo- from the Greek myna meaning nasal secretion

or slime, and hence mucus, was particularly apt.

The development of phosphotungstenic acid negative staining

showed all the myxoviruses to be morphologically similar, consist-

ing of a coiled helix surrounded by an envelope covered with spike-

like projections. At about the same time the group as a whole

were also shown to be RNA viruses. However, measles, distemper,

rinderpest and respiratory syncytial viruses were all shown to be

similar in morphology possessing spiked envelopes and helical RNP,

and although only measles showed haemagglutination activity and

none possessed nouraminidase they were added to the group (Virus

Sub-Committee of the International Nomenclature Committee, 1963).

Lwoff, Horne and Tournier (1962) proposed that viruses should be

classified by the following characteristics 1) Nucleic acid,

DNA or RNA?; 2) Symmetry of nucleocapsid; 3) Presence or

absence of an envelope surrounding the nucleocapsid; 4) The

diameter of the nucleocapsid or the number of capsomers. The

myxovirus group were therefore: 1) RNA 2) Helical 3) Envel-

oped. But on the basis of the diameter of the nucleocapsid the

myxovlruses could be divided into two groups (Waterson, 1962):

The influenza group with an inner helix diameter of 9-10 mm,

including the influenzas and fowl plague. The NDV group including

NDV, mumps, the parainfluenzas, measles, rinderpest and distemper virlAse-%

all of which have a helix diameter of 17-18 nm. (Fig. 1)

This method of classification presents some problems however as

viruses such as rabies would fall into one or other of the groups

although morphologically as well as in other respects they are

fundamentally different.

It has since been suggested (Waterson and Almeida, 1966) that

a further distinction of more significance than the size of the

nucleocapsid should be made in the myxovirus group.- This is to

separate those viruses showing an affinity fsti'mucoproteins, i.e.

the myxophilic 'true myxoviruses' which possess neuraminidase and

those which do not, "pseudomyxoviruses". The scheme suggested by

Waterson and Almeida is outlined in Figures 1 and 2 . In the

original scheme bovine parainfluenza 3 virus was classified as a

pseudomyxovirus of NDV morphology but it has since been shown to

possess neuraminidase (Drzenialt,, B8gel and Rott, 1967) and so

should be included in the true myxovirus group.

9

FIGURE

The Lwoff-Horna•Tournier Classification of Myxovirusos.

10

Virus

RNA

Helical

Enveloped

170-180 a

NOV Rabios 90A

Mumps VSV

Parsinfluanzas Avion orythro-

Measles -myeloblastosis 42A

Rindarpott

DistanTer

Characteristic:

Nucleic acids

Symmetry:

Envelops{

()isolator of nuclaocapsid:

O

90-100 A

InfIvonzas

Fowl plague

FIGURE 2

The OT-thitarson-Almoida Classification of Myxoviruaeo.

11

Characteristic:

Nucleic acid:

Symmetry:

Envelopes

Myxovirus morphology

Virus

RNA

F-- Helical

F-- Enveloped

Uthor compound helical viruses so. Rabies, Viltit Avian orythromyolo--blastosis

Myxophily:

True myxoviruses

Size of nucloocapsids

influon2se NOV

Pseudomyxoviruses

'NOV morphology' 'Influmorpenza hology'

1(11s1as •••

ParninflunnmOindorpnnt

Diatnmpor

D. REPLICATION OF NDV

In many instances the details of NDV replication are not Fo be-

specifically known and are assumed/analogous to other myxoviruses.

In the following account, unless otherwise specified, all ref-

erences are to experiments with NDV.

1. Adsorption

Presumably adsorption of NDV to cells is mediated by haema-

gglutinin attachment to specific cell sites. After treatment of

cells with Vibrio 'cholerae neuraminidase cells fail tolfuse, become

infected, and red cells fail to agglutinate.on inoculation with

NDV, because of the destruction of the cell-receptors. (Kohn, 1964,

Waterson, 1968).

The actual mode of attachment seems to be controlled by

electrostatic forces for while it is impossible to adsorb virus

particles in isotonic glucose medium free,. of ions, adsorption can

he achieved by the addition of electrolytes, divalent cations

being the most efficient (Rott and Scholtissek, 1967).

2. Penetration

&makes de St. Groth (1948) proposed that myxoviruses entered

host cells by active engulfment (viropexis) of whole virus by the

cells. Musegay and Weibel (1962) enhanced this theory by showing

that particles of intact NDV virions can be observed in the cyto-

plasm as early as 30 minutes after inoculation of chick embryo

cells. It is postulated that liberation of RNP then occurs. Hoyle

(1962) has shown in in vitro studies that normal cytoplasmic par-

ticles of the chorioallantoic membrane engulf virus particles

causing disintegration and release of RN?. This release of RNP

12

has been shown to occur in cells with 32

P labelled influenza virus

(Hoyle and Frisch-Niggemeyer, 1955). The release of the RNP and

the uncoating of the viral RNA all appear to be carried out by

enzymes associated with lysosomes (Ralph, 1969).

3. Eclipse Phase

a) General Events

Because NDV cuts off cell macromolecule metabolism only very

slowly, if at all, study of many of the events during the eclipse

phase, in particular viral RNA production, would be extremely

difficult if NDV was not relatively insensitive to actinomycin D

(Kingsbury, 1962).

The synthesis of viral RNA starts between 2-4 hours after

infection, reaching a maximum at around 7 hours. The actual site

of RNA replication in the cell is in some dispute. Wheelock (1963)

claimed the cytoplasm was the site of NDV RNA synthesis. However

Bukrinskaya, Burdulea and Vorkunova (1966) point out that unless

the labelled pulse is brief the label is found throughout the

cytoplasm; whereas with short pulses of labelled precursor they

were able to show, by autoradiography, that RNA appears to replicate

in the nucleolus and migrate to the cytoplasm. More recently

Brett and Robinson (1967) have presented further evidence for a

cytoplasmic site, however, they have sampled somewhat later after

infection and may have missed an early nuclear site.

Between 3-4 hours after infection RNP, haemagglutinin and

neuraminidase are produced in the cytoplasm and shortly afterwards

infective virus can be detected.

b) Types of NDV-specified RNA produced during infection

It has been shown that of the RNA produced in NDV infected cells

13

80% of that produced between 3-5 hours and 90% of RNA produced

at 7 hours is complementary (-)RNA (Kingsbury, 1970) although some

(+) RNA is always produced (Kingsbury, 1966b, Brett and Robinson,

1967, Blair and Robinson, 1968). Some of the (+) RNA sediments

at the same rate as RNA from virions (570 but there is also more

slowly sedimenting (+) RNA which may be free or associated with

(-) RNA (Brett and Robinson, 1967). It is not sure in these

experiments however how much (+) RNA is derived from nucleocap-

aid or cell associated virions. Evidence generally suggests that

there is no pool of viral genomes although work by Reeve and Poste

(1970) suggests genome RNA may be used as a messenger.

The NDV specified complementary RNA is heterogenous in sedi-

mentation behaviour producing peaks'at 188, 228 and 358 and some

57S RNA (Brett and Robinson, 1967, Blair and Robinson, 1968).

Brett and Robinson (1967) showed that 50% of the NDV genome

is made RNAse resistant when annealed with (-) 183 RNA. Since 18S

RNA would probably have a molecular weight one sixth of that of

the NDV genome this work suggests that 188 (-)RNA may be hetero-

genous with respect to nucleotide sequence.

c) Role of complementary RNA

In other non-myxoviruses it is generally accepted that com-

plementary RNA functions as a template for (+) RNA production,

the viral strand being replaced in the double-stranded complex as

a new strand is initiated. In NDV infections the most logical can-

didate for this function is a 57S(-)RNA, i.e. one of the same size

as the genome. However this does not account for the vast majority

of the (-) RNA which has insufficient information to fulfil this

function. Similarly although small amounts of 35S and 57S RNAs

14

resist RNase digestion, there is no clear evidence of RNase-

resistant double-stranded viral RNA replication intermediates

(Brett and Robinson, 1967).

An alternative hypothesis is that the (-) RNAs are messenger

RNA. The fact that most of the RNA associated with the polyriba.

somas is of the (-) RNA type supports this hypothesis (Brett and

Robinson, 1967, Blair and Robinson, 1968, Kingsbury, 1970), although

the presence in the polyribosomes may be fortuitous.

Schafer, Pieter and Schneider (1967) by showing viral antigens

are produced in cells even after viral RNA synthesis is inhibited

have proposed that NDV (+) RNA functions as the sole messenger.

Work by Reeve and Poste (1970) has strengthened this opinion while

Ralph (1969) suggests that both (-) RNA and (+) RNA may serve as

messengers at different stages of the infection by NDV.

d) NDV-specified RNA dependent RNA-polymerase

Although it is a fairly obvious speculation that a NDV-

specified RNA-polymerase is produced in infected cells there is

currently only one report of such enzymii activity. The enzyme

detected by Scholtissek and Rott (1969) was not found in noninfected

cells and was not inhibited by actinomycin D. The enzyMe was

first detectable 3 hours after infection, the peak activity was

reached after 5 hours. Predictably it was shown by nearest neighbour

analysis and hybridization experiments that the vast majority if

not all the RNA produced in vitro by the enzyme was (-) RNA.

However no information vas recorded as to the sedimentation coeffic-

ient of this RNA. The RNA polymerase activity was detected in

the cytoplasmic extract of infected cells but there is no indication

as to whether or not the nuclear fraction was tested for activity.

15

4. Virus maturation and release

However much the myxovirus subgroups differ from one another

in other respectsi the maturation of the virion at or near the cell

surface seems to be common to all the myxoviruses (Chanock and

Coates, 1964). For this reason it has been generally accepted

that the mode of release for all the myxoviruses is very similar.

Mature NDV production is detectable about 4 hours after

infection, with cytopathic strains production ceases after 10,42

hours but with non-cytopathic strains virus production may con-

tinue somewhat longer. At about the same time as mature virus is

first detectedi Narcus (1962) has shown that infected cells become W4V-

able to adsorb red cells and suggested/this was brought about by

the incorporation of viral heemagglutinin into the cell membrane

prior to virus release: Furthermore Rott (1964) has shown that

prior to release iviral nucleocapsid can be seen to accumulate

beneath the cell surface of NDV infected cells.

Using SV5 virus (Compans et al, 1966) it has been shorn that

the nucleocapsid becomes aligned under the cell membrane and is

slowly budded off to form a released virus particle, part of the

infected cell membrane becoming the viral envelope. It is not

clear, however, whether the nucleoprotein aligns and then cell sur-

face modification occurs or vice versa.

Seto and Rott (1966) have demonstrated that neuraminidase

plays an important part in fowl plague virus (FPV) release. When

neuraminidase specific antiserum was added to FPV infected mono-

layers no HA, infective virus or neuraminidase activity was

released from the cells, although the normal amount of complement

fixing antigen and at least one third of the normal infectivity

16

was detectable associated with the cells. These findings have been

confirmed morphologically by electron microscopy of infected cells

after treatment with specific antiserum (Seto and Chang, 1969).

It has been estimated that the release time of NDV. from chick

embryo lung epithelium cells is one hour (Rubin, Franklin and

Bamda, 1957).

17

E. VIRULENCE - RANGE, AND METHODS OF ASSESSMENT

Although Newcastle disease has remained endemic in this

country since the 1940's, until the present outbreak, highly vims

lent outbreaks similar to those of the 1920's were rare. Occasional

outbreaks causing high mortality were generally self limiting or

controlled by slaughtering the infected fowls. Because of these

conditions of low virulence endemic virus and occasional virulent

outbreaksi many strains of NDV have now been isolated and they show

a continuous spectrum between the two limits of virulence.

Three methods have been universally accepted for the

assessment of virulence. They are detailed in Methods for the

examination of poultry biologics (1963).

a) Mean death time in embryonated eggs (MDT)

The mean death time is the average time in which eggs inocu-

lated with one minimum lethal dose of virus are killed.

b) Intracerebral pathogenicity in chicks

The intracerebral pathogenicity index (ICPI) is estimated

from time taken for one-day-old chicks to die or show disease symp-

toms after intracerebral inoculation of virus. The results are

based on a scoring system in which the maximum index possible is

2 (100% mortality in one day) and the minimum is 0 (no recorded

symptoms after 8 days).

c) Intravenous pathogenicity index (IVPI)

This is the least used method. It is very similar to the

ICPI method but 6-week-old chicks are used and are injected intra- ,

venously,/maximum score is 3.

Table 1 shows the MDT, ICPI and IVPI of all the strains used

18

TABLE 1

THE VIRULENCE OF NDV STRAINS

STRAIN ICPI* MDT* IVPI** PLAQUINCABILITY IN (Hr) CHICK EMBRYO CELL

CULTURES

Field Pheasant 2.00 54 2.69 +I-

Herts 1.88 49 2.64 ++

Italian 1.86 50 2.81 -H-

Texas 1.75 55 2.66 +I-

Warwick 1.72 62 2.64 -t-i-

Eastwood Notts 1.70 70 1.70 +

Beaudette C 1.46 62 ND +

it." 1.21 48 0.0 ++

B1

0.40 117 ' 0.0 ±

F 0.25 119 0.0

Queensland 0.16 m 0.0

Ulster 0.00 co 0.0

++ Large well-defined plaques

+ Small -till-defined plaques

+ Plaques very small ill-defined often absent

• Does not form plaques

* Source: Alexander, Reeve and Allan, 1970

** Source: Allan - unpublished data

19

in this study. The large range of virulence can be seen. Hanson

and Brandly (1955) grouped the strains of NDV according to the

MDT:-

Vologenic strains MDT 40-60 hours

Nesogenic strains MDT 60-90 hours

Lentogenic strains EDT > 90 hours

The divisions are purely arbitrary but are worth mentioning as the

terminology is still often used.

Other properties can also be used as a guide or a measure of

virulence. For example, Schloer and Hanson (1968) have shown that

plaque size is directly related to virulence. The ability to

plaque at all is also indicative of virulence and the growl diff-

erencesof plaguing ability are also shown in Table 1.

It is worth noting that although the different measures of

virulence run parallel or inversely paral1t there are strains

whose relative virulence varies with the method used. Strain 11

for example shows low virulence in chicks and yet produces the

lowest EDT in eggs and the largest plaques in chick embryo fibroblasts

of all strains tested. This may indicate a difference in the

cause of virulence from one system to another.

20

F. STRAIN DIFFERENCES AND VIRULENCE

Virulence Definition

'The capacity of a micro-organism to kill is covered by the

term virulence, and virulence may be determined by toxigenicity,

invasiveness, or many other factors and is qualified by the host

in which it is measured.' (Wilson and Miles, 1964)

21

Foreword

'In any study of virulence in the NDV system it is particularly

important that the number and range of virulence of the strains

used are carefully selected: In the past often only two strains

have been studied, a virulent and a less virulent. Because of

this strain differences have sometimes been put forward as caus-

ing a difference in virulence but which are merely differences

between the two strains used. It is particularly unfortunate that

in many of the studies Beaudette C has been used as the strain

of lower virulence (Rott, Reda and Schafer, 1962, 1963, Schafer

and Rott, 1959, Drzeniek, Seto and Rott, 1966, Wilson, 1968 etc).

Beaudette C is not an avirulent strain being classed as mesogenic

by Hanson and Brandly (1955) but in fact is considerably virulent

in eggs,(see Table 1). Beaudette C is also a particularly atyp-

ical strain as it is not a natural isolate but a laboratory mutant

isolated solely for its heat stability (Granoff, 1959).

To reach any conclusions as to the cause of the wide range of

virulence in the NDV system it is necessary to study a reasonably

large number of strains which cover a wide range of virulence

from all parts of the spectrum. The converse, however, is not

necessarily true and it is safe to say that any one property is

not the cause of difference in virulence if one virulent and one

avirulent strain are identical in that property, providing the

strains are frankly virulent and frankly avirulent.

22

1) Morphology and virulence

The pleomorphic nature of NDV virus makes morphological

comparisons between strains difficult. Waterson and Cruickshank

(1963) compared the morphologyi before and after ether treatment)

of eight strains of varying virulence. There was some variation,

though of no relation to virulence, in the ease of ether disrup-

tionibut morphologically the whole virion, the diirupted particles,

end the RNP were similar for all the strains tested. Rott, Bade

and Schafer (1962) reported that up to 187. of their preparation

of strain Beaudette C had no RNP while strain Italian preparations

had only 27. empty particles. Since only two strains were examined

further work is necessary to show if there is any relationship

with virulence or not.

2) Serology

Serological studies of the surface antigens of NDV strains

using a serum neutralisation test (Upton, Hanson and Brandly, 1953)

showed up to 105-fold differences between strains. However, the

differences shown in no way related to the virulence of the strains.

The procedure used by Upton et al (1953) was later discredited

and using the now accepted procedure for serum neutralisation reac-

tions and comparing haemagglutination inhibition it has been shown

that no significant differences exist between the most virulent

and avirulent strains. (Waterson, Pennington and Allan, 1967).

Pennington (1967) using more sensitive techniques, and examin-

ing the internal nucleocapsid antigen as well as the external anti-

gens, showed only minor serological differences between strains

which in no way correlated with virulence.

The word strain then, when applied to the NDV system, is used

23

to mean a stable well characterised line of virus, and does not

imply a serologically distinguishable type.

3. Biophysical properties

Strains that differ as widely in virulence as those of HDV

may well differ in the constituents of the virion or in the ratio

of these constituents. The variation may be sufficient to give

a different bouyant density to the virions of the strains.

Roisman and Roan&-.(1961) have shown just such a difference between

two strains of herpes simplex virus. cletd showed different

cytopathic effects but otherwise appeared identical. However,

they could be separated on caesium chloride gradients. [T-he strains

were both grown under identical conditions on the same cell lice]

Stenback and Durand (1963) showed that NDV populations of

strain California behaved as if they contained infectious particles

with a wide range of density. By using a wide range of cells for

virus growth it was shown that the density in caesium chloride

was host controlled. The average densities in various avian cells

was between 1.219-1.221 whereas in mammalian cells 1.236-1.242.

The host control is not totally unexpected as the envelope of

HDV which must greatly affect the bouyant density is generally

accepted as being a modified piece of cell membrane (Cruickshank,

1964). This however raises the possibility that avirulent NDV

strains may have a different density than virulent strains due

to their reported maturation at different sites in the cell

(Bang, 1953).

4. Cytopathogenicity

During NDV infections there are several different kinds of

cytopathic effect and often only one particular effect has been

24

studied by any one author. For this reason this section is

divided into six parts although this is in no way intended to

suggest different mechanisms are involved or that the effects are

not different manifestations of the same event.

a) Haemadsorption

The ability of red cells to adsorb to the surface of infected

cells is the first evidence of cytopathic effect seen in NDV

infections. Marcus (1962) has shown that haemadsorption can be

detected as early as 4-6 hours in his HeLa or chick embryo cell

systems. He suggests that this cell surface modification is

brought about by the incorporation of newly synthesized haemagglu-

tinin into the membrane. Using a system in which single cells

could be studied he show& that, following normaf- growth curve kin-

etics, the amount of haemadsorption increased from a single BBC

per cell until the whole cell surface wts covered by adsorbed red

blood cells. Very little work has been carried out comparing the

ability of strains of NDV to induce the phenomenon but Bankowski

(1964) him shown that the less virulent strains tend to take longer

to show haemadsorption in monolayers.

b) Syncytial formation - cell fusion from within

When cells are infected with a cytopathic strain of NDV,

syncytia can first be noticed at about eight hours after infection

(Brett and Gallaher, 1969) and gradually increase in size, as well

as in numbers, by the fusion of more cells until cell death occurs.

There is no evidence that the size of single syncytia which may

contain 3-200 or more nuclei or the overall formation in a monolayer

has any relationship with strain virulence. However it has been

25

shown that the avirulent strain B1 fails to form syncytia

(Bankowski, 1964).

c) Plaque formation

If low doses of virus are applied to a cell sheeti between

24-72 hours/discrete:lesions (plaques) can be seen in the sheets

particularly after staining with neutral red. Neutral red is

adsorbed into the lysosomes of active cells hence normal cells

appear red in colour while dead or dying cells fail to take up

neutral red. Not all strains of NDV are capable of forming plaques.

Under certain conditions it has been noticed, with viruses

other than NDV, that groups of infected cells take up more neutral

red than surrounding uninfected cells and give the appearance of

'negative' or 'red' plaques (Bonifas and Schlesinger, 1959). Thiry

(1963) showed that similar red-plaque-forming mutants occurred

spontaneously in wild-type NDV populations and could be induced

using nitrous acid or hydroxylamine as mutagens. Thiry further

showed that cell sheets recovered completely from infections by

red plaque mutants but were destroyed by white mutants.

Bankowski (1964) has studied the cytopathology of five strains

of NDV of differing virulence and recorded the cytopathic occur-

ences over 72 hours, his work is summarised in Table 2.

d) Plaque size

Schloer and Hanson (1968) showed that of the 14 representa-

tive strains of NDV they studied only the velogenic and mesogenic *rains

formed plaques. Furthermore it was shown that of the strains that

formed plaques i the frankly virulent strains produced much larger

plaques than the mesogenic strains, and, as a general trend the

larger plaques were formed by the more virulent strains. Kohn and

26

TIME OBSERVED HOURS p.i.

TABLE 2

CYTOPATHIC EFFECTS OF DIFFERENT STRAINS OF NDV

SOURCE: BANKOUSKI (1964)

CPE/STRAINS

TEXAS

CAL 11914

TCND

A

B1

2

6

I0

24

48

72

•••

•••

WO

OM. IMO

Small syncytid formed, showing haemadsorption

Cell sheet destroyed

Cell sheet destroyed

As for Texas

Large syncytia and massive haemadsorption at edges of discrete plaques

As for Texas

As for Texas

As for CAL 11914

As for Texas

Very small syncytia and microscopic plaques some haem-adsorption

Destroyed 501 of cell sheet. Remaining cells show evidence of small plaques

Some cell death and occasional red cell attachment but no syncytid or plaques even when very high multiplicities used

Cells: Chick embryo

Inoculum: 100 TCID 50 per Leighton tube

Fuchs (1969) however, using eight strains of NDV were unable to

show more than a general tendency for plaque size to vary with

virulence. There is though no doubt that the very virulent strains

produce largo well defined plaques, the more moderately virulent

strains smaller, less well-defined plaques and the avirulent strains

no plaque at all.

e) Lysosome damage

Allison and Sande cin(1963) suggested that in virus infections

release of lysosomal enzymes may well contribute to cytopathic

effect. Further work, with viruses which varied in their cytopathic

effect including red and white plaque mutants of NDV, (Allison

and Mallucci, 1965) has shown that there are apparently two stages

in lysosomal damage:

i) Lysosomal membranes become permeable but do not Nr

release enzymes. This is demonstrable by the increased

uptake of neutral red into the lysosomes giving rise to

red plaques. The cell may recover from this state as it

does with the red plaque mutants of NDV.

ii) Lysosomal enzymes are released into the cytoplasm

due to further increase in permeability of the membranes.

The cells round up and uptake of neutral red decreases,

resulting in the formation of white plaques.

Work with other viruses has tended to confirm these original

findings (Allison, 1967, Poste, 1910) and lysosome damage is now

considered as almost certainly a cause of cell fusion if not total

cytopathogenicity.

f) Syncytial formation - cell fusion from without

If animal cells are treated with large concentrations (> 100

28

KID50/cell) of NDV, cell fusion occurs and polykaryocytes contain-

ing 3-150 nuclei/cell may be formed (Kohn, 1965). The fusion

begins within an hour and reaches a maximum within 2-4 hours. The

extent of cell fusion varies with the input multiplicity. Brett

and Gallaher (1969) have shown that this is a quite separate phen-

omenon from that of cell fusion after infection (fusion from

within). Fusion from within requires low inoculli of infectious

virus and protein synthesis while fusion from without needs neither

infectious virus nor protein synthesis for induction. They have

also shown that strains of NDV which induce fusion from within

are not necessarily good at inducing fusion from without and that

strains that are particularly good at fusion from without do not

necessarily produce fusion by infection at all.

Kohn (1965) attempted to demonstrate the viral component

responsible for fusion from without. Destruction of the cell recep-

tors with Vibrio &holerae neuraminidase stopped cell fusion so it

is necessary for the virus to become attached to the cells. However

since phospholipases were the most active in destroying cell fusion

ability Kohn concluded that the integrity of the phospholipids of

the viral membrane was the main requirement for the formation of

polykaryocytee.

Kohn and Fuchs (1969) studied eight strains of NDV and showed

that the ability to fuse cells from without varied enormously from

strain to strain but there was no correlation whatsoever with this

ability and virulence.

29

5) Biological Activities

In addition to cytopathogenicity NDV viriona have several

other biological activities associated with them: haemagglutina-

tion,neuraminidase, haemolysin, ADPase and ATPase. It is a

short step to argue that the differences in virulence may be due

to greater or smaller amounts, or greater or lesser activity of

one or more of these active units.

Only HA and neuraminidase of the activities listed above

appear to be integral parts of the virus structure; the others

being of host cell origin, perhaps derived from cellular lysosomes

(Neurath, 1964, Allison, 1967). However, Kohn and Fuchs (1969)

have shown that the variation in haemolysin activity has no rela-

tionship with the variation of virulence of the eight strains they

tested.

a) Haemagglutinin

Although the evidence is somewhat confused it appears that

viral haemagglutinin activity is in no way related to",,virulence.

Studies with unpurified virus are limited in their use since Rott,

Reda and Schafer (1962, 1963) have shown that with Beaudette 'C 187.

of HA present in their preparations was due to empty virus particles

whereas with Italien the figure was only 2%. Purification is

evidently necessary so that intact vArions only are examined.

Kohn and Fuchs (1969) using 8 strains of purified virus showed that

although the ratio of HA to infectivity varied it did not correlate

with the differences in virulence. Schafer and Rott (1959) reported-

that treatment of virus with ether produced a large fall in HA

activity with strain Italien whereas Beaudette C retained its

activity after treatment. Pennington (1967) examined many more

30

strains and showed that the loss or retention of HA activity after

treatment with ether is independent of virulence. Kohn and Fuchs

(1969) have also shown that the differences in heat lability of

HA is not related to virulence.

b) Neuraminidase

Of all the activities associated with the virion the most

likely candidate to cause the differences in virulence is neura-.

minidase. It seems highly likely that the production of neuramini-

dase in any quantity in the cell would lead to cell damage due to

the effect - on the mucoproteins in the cell membranes, Scholtissek,

Becht and Drzeniek (1967) have in fact shown that the cytopatho-

genicity of influenza virus was related to the presence of neura-

minidase activity.

Drzeniek, Seto and Rott (1966) have shown that the neuramtni-

dases of Italian and Beaudette C are identical in serological and

chemical properties, these strains are close in virulence but assum-

ing their findings applied to all strains then any variation of

neuraminidasa activity that correlated with virulence_ would be

quantitative rather than qualitative. Mussgay (1960) comparing

strains Italian (virulent) and B1 (avirulent) showed that the rate

of elution from fowl erythrocytes was far greater with Italian.

Howevar,it is difficult to make any conclueiona as to the relation-

ship with virulence from an experiment using only two strains.

It should be borne in mind that even if the quantity or activity

of the virus subunits do not vary from strain to strain as part of

the virion, the intracellular production may be unrelated to the

amount per virion. The possibility of this is discussed elsewhere

in this review (see virus multiplication growth studies section).

31

6. Virus multiplication

a) Growth rates

In any study with growth rates and virulence it is important

that 'one-step' growth curves should be examined. Using a one

step growth curve secondary adsorption, infection and inactivation

will not necessarily effect the experiment and that in fact the

growth cycle in one cell is being measured although with many

duplications. It is also important that both intracellular and

extracellUlar (or released) virus is measured as cell death may

be due to failure to release virus or due to damage at virus

release. For example Compans et al (1966) showed that BHK21-F

cells infected with' SV5 virus accumulated viral ribonucleoprotein

and disintegrated after extensive fusion although little mature

virus Was released. In contrast primary rhesus monkey kidney cells

infected with SV5 did not accumulate RNP, released infective virus,

but were not destroyed.

Studies with NDV have usually been confined to two strains,

one virulent and one avirulent. Liii and Bang (1953) compared the

growth of a virulent strain (CG179) and the lentogenic B1 in the

allantois of fertile eggs. The released virus reached a titre of

109 rt,50/mtin 26-28 hours with both strains but a further 10-fold

increase took 56 houri with the vaccine strain and only seven with

the virulent. Drain (1969) found similar results with B1 and Texas

(sea Table 3) but in neither of these cases were one step growth

curves used.

Using chick-embryo fibroblast monolayers and 100 /050/cell (i.e.

one step) Drain 1969 showed that in both intracellular and extracell-

ular production strains Texas and B1 were vary similar but events

32

TABLE 3

GROWTH OF VIRUS IN EGGS

Release of infectious virus into the allantoic cavity after low

infection (100 ID50 per egg)

Titre attained

Time taken (Hrs) ID50/m1

8 3 . 10

3 9. 10

9 8 . 10

10 9

1 10 0

Texas

24-25

35

47

CG 179

26

33

B1

28

40

72

B1

28

84

:Drain (19690

Liu S. Bang (1953)

6)9

with 81 tended to occur later (approximately one hour behind). In

a similar system Reeve and Waterson (1970) showed that the aviru-

lent strains Queensland and Ulster had growth cycles which showed

only minor differences to that of Italian and concluded that the

gross differences in cytopathogenicity between the two avirulent

and virulent Italian were in no way related to the differences in

the kinetics of growth in fibroblasts.

Hirsagay (1960) had earlier made the opposite concluaions

after studying Italian and F, reporting that the difference in

cytopathogenicity of the two strains was probably due to the slower

release of strain F. The differences noted by Mussgay were small,

however, and seem unlikely to account for the large difference in

the plaque sire. The crucial point is whether or not the avirulent

viruses produce plaques when a similar titre of virus to the viru-

lent strains is reached. Pennington (1967) studied strains F,

Queensland and Ulster which did not produce plaques in his system:

even after six days there was no sign of plaques although the titre

of released virus exceeded 10 10*ID50/ml which was greater than

the titre of released virus with virulent strains at the time

plaques were apparent.

The distinction,between those that form plaques and those

that do not is an important one and leads to speculation that there

are two groups which vary in virulence but by- different mechanisms:

1) The plaque formers which within themselves vary in

virulence due to the speed of growth but which eventually all

kill eggs and chickens and which if left a sufficient time would

(it Is postulated) all have a similarlmaximum plaque size.

2) The non-plaque formers which do not form plaques at all

34

and do not differ merely in growth rate.

It is important to remember in the study of growth rates that

with some strains up to 18% of released virus (as measured by

BA) may be present as non-infective virus (Rott et al, 1963) it is

therefore imperative that HA should be measured in conjunction

with infectivity. Fortunately the strains discussed above have

similar levels of non-infective particles.

Pennington (1967) showed that 40 hours after infection the

ratio of the HA titre of homogenated CAM to the HA titre of the

allantoic fluid was much higher for virulent strains than avirulent

strains; eight strains of varying virulence were tested and a

marked correlation was evident between the ratios and the MDT

in eggs. Reeve, Rosemblum and Alexander (1970) have made a more

detailed study of growth of different strains in eggs. Growth

curve studies of both CAM homogenate and allantoic fluid of infected

eggs led them to suggest that there were two discrete groups of

strains: 1) Virulent strains which kill chick embryos rapidly

and accumulate all the major viral components in the chorioallantoic

membrane to titres higher than in the allantoic fluid, and 2) aviru-

lent strains which kill embryos slowly or not at all and produce

similar extracellular antigen titres to virulent strains but in

which the intracellular accumulation of virus antigen is limited.

b) Growth site

The actual site of multiplication has also been implicated in

the differences in virulence of NDV strains. Kaman and Bang (1951)

compared the rates of increase of two strains CG179 (virulent) and

B (avirulent) in various tissues after intramuscular inoculation of

chickens. Both strains increased at the same rate in the external

9 r J

tissues, i.e. the blood, lungs, rectum and spleen, but the viru-

lent strain showed an accelerated growth rate in the brain. The

titre of the virulent strain was in fact 2000 times greater than

that of the avirulent strain after 24 hours. However the aviru-

lent strain eventually reached a higher titre in the brain though

much later. There was no brain damage or chicken death with the

avirulent virus which suggests that speed of growth, rather than

the titre reached is significant.

Bang (1953) also showed differences in the site of growth on

a cellular level by electron microscopy on thin-sections of infec-

ted Chorio-allantoic membranes. The avirulent strain (B1) showed

no damage to the cells and mature virus particles appeared to be

released from microvilli formed at the cell surface. The virulent

strain (C0179) however caused gross cellular damage and disorder

and appeared to mature within the cytoplasm. No other evidence

has been recorded to confirm the suggestion by Bang that this may

be the direct cause of the difference in virulence.

7. Effects on cell metabolism

Many viruses Which cause microscopically or grossly visible

cytopathic effects also inhibit the synthesis of cellular macro-

molecules (Martin and Kerr, 1968). The effects and causes of the

cut off of cell macromolecule synthesis have been studied in detail

for many viruses and although Bablanian, Eggers and'Tamm (1965)

have shown that poliovirus-induced switch-off of cell metabolism

was not responsible for the cytopathic effect of that virus it is

not certain to what extent other virus systems are affected.

36

a) Protein and RNA synthesis

Protein and RNA synthesis are closely linked and in general

have been examined together in studies involving NDV. Inhibition

of cell protein synthesis and RNA synthesis by NDV, using the

virulent Hickman strain, was shown by Wheelock and Tem (1961).

Using HeLa cells it was shown that 53k hours after infection

there was no incorporation of radioactive labelled precursors into

proteins. RNA synthesis appears to be more gradually inhibited;

by hour 10 a decrease of 40% of precursor incorporation had occurred.

One important feature of this work was that protein synthesis cut-

off occurred long before the gross cytopathic effect was apparent,

showing that it was unlikely that cell protein synthesis was

affected merely as a result of cell damage as Rott (personal communi-

cation) has suggested.

Bolognesi and Wilson (1966) using strain Texas G.B. with chick

embryo fibroblasts showed that cell protein synthesis inhibition

was initiated five hours after infection and by nine hours had

reached 85% inhibition.iiing the RNA inhibitor azauridine viral

protein synthesis cut-off could be prevented if the inhibitor was

added at the time of infection but not if added after three hours.

These, and other results with actinomycin D and cycloheximide indi-

cated that proteins synthesised during early NDV replication were

responsible for the inhibition of protein synthesis and the synthe-

sis of these proteins required the prior synthesis of RNA.

Scholtissek and Rott(1965)reported that with strain Italien and

chick embryo fibroblasts inhibition of cell protein synthesis was

reversible and infected cells recovered after ten hours. Wilson

37

(1962) using strains Texas G.B. and Beaudetta C was unable to

show a similar recovery.

Wilson (1962), using Texas G.B. (virulent) and Beaudette C

(mesogenic, - but see note at beginning of section) has made the

only attempt to compare two strains of differing virulence to see

if their ability to cut off cell protein and RNA synthesis varied.

Both strains inhibited protein synthesis to a similar level but

experiments with metabolic inhibitors suggested that different

mechanisms were involved. RNA synthesis inhibition by Texas was

marked (more than 807. inhibition at 10 hours) and shown by use

of cycloheximide and azauridine to involve the synthesis of inhibi-

tory proteins coded by the virus genome. Beaudette C shoved very

little inhibition of RNA synthesis at all, in fact a slight increase

was recorded presumably due to viral RNA synthesis. This however

may not be associated with virulence as Scholtissek and Rott (1965)

showed that strain Italian (highly virulent) produced a similar

effect in their system.

b) DNA synthesis

Wheelock and Teimn (1961) showed that using the virulent

Hickman strain in a HeLa cell system DNA synthesis became almost

completely inhibited by NDV within five hours of infection. Further

work with synchronized and unsynchronized,L cell cultures using

strain Hickman has lead to a greater understanding of the mechan-

ism of DNA inhibition by NDV (Ensminger and TamM, 1970a, 1970b).

Cellular template DNA retains its structure after NDV infection

so that it is unlikely that degradation of template DNA causes

DNA synthesis inhibition. Similarly synthesis and polymerization

enzymes of precursor deoxyribonucleotides are unaffected. However

38

it has been shown that maintenance of DNA synthesis requires con-

tinual protein synthesis in uninfected cells (Ensminger and Tamm,

1970b). Ensminger and Tamm postulate, therefore, that NDV inhibits

cellular DNA synthesis secondary to the inhibition of protein

synthesis and that viral inhibition of protein synthesis affects

a process required for the initiation of DNA synthesis upon new

sites of replication.

c) Lipid' synthesis

Drain (1969) extensively studied the effect on 14C-acetate

incorporation into lipid of infection by strains Texas and B1

(avirulent). Very little difference was observed, both strains

produced gradual suppression of lipid metabolism to as low as

309. by 20 hours after infection.

8. Interferon and virulence

a) Interferon sensitivity

Sensitivity to interferon could obviously be a factor affecting

the virulence of a virus, and it is known that in some virus systems

the less virulent strains are more sensitive to interferon than the

virulent strains (Penner, 1968). However, Baron (1964) has shown

that nine strains of NDV of widely varying virulence are relatively

insensitive to interferon and that interferon sensitivity is unlikely

to have much significance in the variation of virulence between

NDV strains.

b) Interferon production

It has been shown that in cells infected with Semliki Forest

virus (Pinter, 1964), vesicular stomatitis virus (Wagner et al, 1963),

foot and mouth disease virus (Sellers, 1963, 1964) and herpes simp-

39

lex virus (Aurelian and &Oxman, 1965) in the system studied the

lower the virulence the greater the yield of interferon. Fenner

(1968) has suggested that the lack of interferon production in

virulent strains is due to the rapid cut off of cell macromolecule

synthesis by the virulent strains compared to.the'less virulent

,strains. Gandhi and Burke (1970) drew similar conclusions from

' their work with NDV. However Thacore and YOungner (1970) using

strain Harts and a mutant from persistently - infected L cells

showed that in L cells interferon production still occurs although

shut off of cell synthesis is far more complete than in chick

'embryo cells which do not produce interferon on'infection. They

suggest that the defect in induction of interferon synthesis in

chick embryo cells infected with Harts strain of NDV is not rela-

ted to the general shut off of host cell synthesis but to the fail-

ure of a more specific event. Thiry (1963) has shown using 'red'

mutants that in the NDV system the interferon production of cells

in vitro is inversely proportional to the virulence of the infec-

ting strain. Baron (1964) has similar findings with infected

fertile chicken eggs but shows that little if any interferon is

produced with the truly lentogenic strains B1 and LaSota.

Lomniczi (1970a) has studied the in vivo production of inter-

feron in chicks infected with NDV and has shown that virulent

strains are more potent inducers of interferon than avirulent

strains, these findings in fact agree with those of Baron (1964)

as the avirulent strains used were Blb LaSota and F. In addition

Lomnicsi has shown that induction of maximum interferon was uni-

formly at the second hour after infection with avirulent strains

40

whereas with virulent strains the maximum was considerably later

and varied with strain. Looniest (1970b) also showed a different

mechanism was involved,since damage to the viral nucleic acid

affected the interferon inducing ability of the virulent strains

whereas damage to the viral protein affected the interferon induc-

ing ability of the avirulent strains.

41

G. EFFECT OF CONGO RED ON NDV INFECTIONS

Finkelstein (1961) showed that congo red, trypan blue and

other azo dyes protected eggs against low but lethal doses of viru-

lent NDV. Further to this Becht and Dreeniek (1960 have shown

that neuraminidase activity in vitro is inhibited by congo red and

that no infective virus is released from infected chick embryo

cells in the presence of this dye.

Since it has been shown that neuraminidase plays an important

part in myxovirus release (Seto and Rott, 1966, Seto and Chang,

1969; discussed above) it seems likely that Finkelstein's observa-

tions were due to the azo dye inhibiting neuraminidase which pre-

vented release of infective particles. This in turn would mean

that only a sublethal number of cells in the CAH were infected and

subsequently killed.

If congo red does act merely baj prevention of release then,

if sufficiently high doses are used, virulent virus should be unaff-

ected in lethality to eggs.

It has been suggested (Reeve et al, 1970) that the accumula-

tion of virus antigens in infected cells results in the death of

these cells and eventually the death of the infected egg. If

congo red prevents virus release but not virus production in infec-

ted cells it could be postulated that avirulent viruses would

accumulate in and therefore kill cells treated with congo red.

Similarly very high doses of avirulent strains would be lethal to

eggs in the presence of congo red.

42

H. VIRUS-SPECIFIED PROTEINS IN NDV-INFECTED CELLS

When a virus particle infects a cell several events may take

place: uncoating, cessation of cell metabolism, synthesis of

viral nucleic acid, synthesis of virus structural proteins, assem-

bly of proteins,.cytopathic effect and, with NDV, cell fusion and

haemadsorption. Any one of these may require the synthesis of a

separate virus-specified enzyme or other non structural proteins.

A method for investigating the proteins synthesised during

viral infections was reported by Summers, Maisel and Darnall (1965).

This method consisted of radioactive labelling infected cells with

an amino acid in the presence of actinomycin D, to prevent cell

protein synthesis, extracting the proteins using sodium dodecyl

sulphate, and separating them by polYacrylamide disc electrophor-

esis. In this way the proteins of the cell are separated accord-

ing to their molecular Weights and form discrete bands on the gel.

The,gel was then sliced and the radioactivity present in each band -

measured. By this method Summers at al (1965) have shown that 14

virus-specified proteins are made after infection with poliovirus,

while only 4 are seen if pure virus is extracted and similarly

treated. 'Hay, Skehel and Burke (1968) have modified this method

so that host Cell metabolism need not necessarily be stopped. In

their method infected cells are labelled with 3H-amino acid while

uninfected cells are labelled with 14C-amino acid, the uninfected

and infected cultures are harvested, mixed and then, after extrac-

tion and disc electrophoresis, sliced and counted. The ratio of

the 3H dpm to the 14C dpm is highest in bands which are virus-

43

specified since these proteins are not present in uninfected cells.

Hay at al (1968) have shown by this method that Semliki Forest

virus grown in chick embryo cell monolayers formed six virus-

specific proteins, only two of which were present in similarly

extracted pure virus. This refinement of the method is partic-

ularly useful for studies with NDV, because although NDV is insen-

sitive to actinomycin D added after infection it has been reported

to be affected by the addition of actinomycin D to cultures before

infection (Kingsbury, 1962, Granoff and Kingsbury, 1964, Brett

and Robinson, 1967). Addition of actinomycin D at the time of

infection would mean that analysis of virus-specified proteins

produced immediately after infection would be masked by cellular

protein synthesis still occurring, unless the double labelling

technique is used.

Virus-specified proteins may be of great importance in the

NDV system since it can be postulated that one particular protein

(or proteins) which is responsible for cytopathogenicity and cell

death is present only in virulent strains. The time at which

specific proteins are produced during NDV infections can also be

monitored by this method and may prove significant to the virulence

of the infecting strain.

44

3. PURIFICATION OF NDV

Purification of NDV, and large RNA viruses in general, poses

several special problems, mainly because the virions have many

physical and chemical properties similar to those of cellular mem-

branes and because of the great range in size of individual par-

ticles.

Adsorption and elution from erythrocytes (Ada and Perry, 1956,

Hoyle, Horne and Waterson, 1961) has been used for myzovirus puri-

fication but the main disadvantage of this procedure is the intro-

duction of red cell debris into the virus preparation.

The surface properties of myzoviruses have also been used to

separate and purify virus by column chromatography using, alumin..

ium phosphate-silica gel (Prommhagen and Knight, 1959), calcium

phosphate (Taverns, Marshall and Fulton, 1958), or ion exchange

cellulose derivatives (Wilson, 1962, Nicoll, Retail and Colobert,

1964).

Commonly, virus is partially purified and concentrated by

differential centrifugation, consisting basically of low speed

centrifugation to remove large fragments of cellular material and

high speed centrifugation to collect the virus. However signifi-

cant amounts of cellular material remain in the virus pellet. •

Ammonium sulphate precipitation has also been used as an initial ,

concentration step (Minocha, Consign and Eisenstark, 1968) but is

less desirable since it is known to cause loss of infection in sev-

eral viruses (Robinson and Duesberg, 1968).

Centrifugation in density gradients separates components on

the basis of their buoyant density (isopycnic separation) or rate

45

of migration which may be determined by size and shape (rate zonal

separation). Gradients of heavy metal salts such as caesium chlor-

ide have been used to purify NDV (Minocha et al, 1968, Stenback and

Durand, 1963) but with high loss of infectivity. Potassium tar-

trate (McCrea, Epstein and Barry, 1961) and sucrose (Duesberg and

Robinson, 1963) gradients have produced far superior yields of infec-

tious virus.

The introduction of specially designed 'zonal' rotors has

enabled large volumes of viral suspension to be purified to a high

level by a fairly simple method (Fox at al, 1967, Fox et al, 1968).

The major disadvantage of density gradient centrifugation is

that the buoyant density of the myxoviruses range from 1.15-1.25

g/ml which is exactly the range of density of most cell membranes.

This may result in difficulties in purifying highly contaminated

suspensions of tissue culture-grown virus.

In this study due to the availability of zonal rotorsi density

gradient centrifugation was chosen for the purification of virus.

Most of the virus to be purifiedkis egg grown and does not suffer

from gross contamination with cell debris.

46

K. OBJECTS OF INVESTIGATION

It is the object of this investigation to obtain a fuller

understanding of the broad spectrum of virulence seen with diff-

erent strains of NDV.

The major part of this work deals with the proteins of NDV:

the structural proteins, their activity, quantity and the number

present in the virion; an investigation into the virus-specified

proteins and the time that they are produced.

An attempt has also been made to clarify past results which

have been confusing or contradictory. Consequently the phenomena

of cell fusion and haemadaorption, cell protein synthesis inhibi-

tion and the growth of strains of NDV in eggs and cell culture

under certain conditions have all been studied with respect to

variation in strain virulence.

47

II MATERIALS AND METHODS

A. REAGENTS AND EQUIPMENT

1) Reagents

The following materials were obtained from the manufacturers

specified below. A11 other materials wore normal laboratory reag-

ents of 'Analar' quality unless otherwise stated.

Congo Red (for microscopical staining); sodium dodecylsulphate

(specially pure): B.D.H. Chemicals Ltd., Poole, England.

Actinomycin D (STAT..PACK); Nauramine Lactose (from beef colos-

trum): Cal -biochem Ltd., Los Angeles, U.S.A.

Acridine Orange, Gismos Solution, May Grftwald solution, Tween

20; G.T. Gurr Ltd., London England

Buffer Tablets pH6.8: Hopkins and Williams Ltd., Chadvell Heath,

England.

Crystamycin (Sodium penicillin G + streptomycin): Glaxo Labora-

tories Ltd., Greenford, England.

Trypsin solution (sterile, freeze-dried); 4.4% sodium bicarbon-

ate; Eagle's HER medium 10 x strength; Tryptose phosphate broth:

Wellcome Reagents Ltd., Beckenham, England.

Foetal calf serum; Flow Laboratories Ltd., Irvine, Scotland.

Ianagar No. 2; Earle solution (Earle's saline): Oxoid Ltd.,

London, England.

Sephadex G-200: Pharmacia, Uppsala, Sweden.

The following radioisotopes were all obtained from The Radio-

chemical Centre, Amersham, England:

L-Leucine 4,5-T; 19.7-23.0 C/MM (3H-Leucine)

48

L-Leucine-C14(u) 344mC/MM (14C-leucine)

Utidine -5 -T, 17.5-26.5 C/mM (311-uridine)

D -Clucosamine-1 -C14 hydrochloride, 45mC/mM (14C-glucosamine)

L-Arginine -5 -T monohydrochloride, 500 mC/mM (3H-arginine)

n-Hexadecane 1,2, -H3, 200pC/g (3H-hexadecane)

n -Hexadocane -1 -C14 1.10pC/g (14C -hexadecane)

Fetuin

Fetuin was prepared from foetal calf serum by the method of

Graham (1961).

Phosphate Buffered Saline (PBS)

PBS was made from tablets of phosphate buffered saline 'A'

(Dulbecco and Vogt, 1954) obtained from Oxoid Ltd., London.

Coll Culture Media

i) Growth medium

Growth medium was prepared with sterile distilled water to

contain:

10% v/v Eagle's BHIC 10 x strength

10% v/v Foetal calf serum

2% v/v Sodium bicarbonate (4.4% solution)

107. v/v Tryptose phosphate broth

Penicillin and streptomycin to a final concentration of 200

units and 200pg respectively.

ii) Maintenance medium

Maintenance medium was prepared in the same way as growth

medium with the following exceptions.

Foetal calf serum 2%

Tryptose phosphate broth 5%

49

Chicken Red Blood Cells

Chickens ware bled from a wing vein into Alsever's solution

(sodium citrate 2.25g, citric acid 0.8g, glucose 2.2g and water to

100 ml.). The blood cells were then washed three times and suspen-

ded in PBS. The 'packed cell' concentration was calculated using an

haematocrit tube, centrifuging at 3,000 r.p.m. for 20 minuntes in

a M.S.E. 'minor' centrifuge.

2) Equipment

a) Centrifuges

The following centrifuges were used: M.S.E. superapeed 65,

M.S.E. 'minor', M.S.E. 6L; Measuring and Scientific Equip-

ment Ltd., London, England. Sorvall superspeed RC2-B, Ivan Sorvall

Inc., Conneticut, U.S.A.

b) Spectrophotometers

The following spectrophotometers were used for Optical density

measurements: SP600 series 2, SP500 series 2, SP8000; Unice&

Instruments Ltd., Cambridge, England.

LKB Uvicord /I; LKB Produkter AB, Stockholm, Sweden.

c) Densitometers

Photographs of stained gels were scanned using the SP590

electrophoresis strip scanning accessory to the SP500.

Unstained and stained gels were scanned,sed-using the gel-

scanning attachment to a Hilger-Gilford Reaction Kinetics Spectro-

photometer.

d) Fraction collector

In all experiments involving fraction collecting an Ultrorac

7000 automatic fraction collector (LKB Produkter AB, Stockholm,

Sweden) was used.

n Ju

a) Microscopes

A Leitz Orthoplan microscope (Ernst Leitz, Wetzlar, Germany)

was used for most microscopy and photomicroscopy.

A Nikon inverted microscope (PEN Optics Ltd, London, England)

was used for cell culture work.

51

B. VIRUS STRAINS AND VIRUS TECHNIgpES

1) Strains

The following strains were used in this investigation, their

virulence for chickens, chick embryos and ability to form plaques in

chick embryo cell monolayers is given in Table 1.

Harts 33 (Herts)*, isolated at Weybridge in 1933 (Dobson, 1939).

Field Pheasant*, isolated at Weybridge in 1962 from pheasants,

(Allan, personal communication).

Texas C.B. (Texas)* isolated in 1948 near Austen, Texas, U.S.A.

(Boney, 1951).

Warwick* isolated in Warwickshire in 1966 (Allan, personal

communication).

Italian**, isolated in Italy at some date before 1949 (Schafer,

Schramm and Traub, 1949).

Eastwood Notts*, isolated in England in 1966 (Allan, personal

communication).

Beaudette C**, this strain is a heat stable variant isolated

from the Beaudette strain of NDV by Granoff (1959). The parent

strain was isolated in the U.S.A. before 1946 (Granoff, 1964).

'11 1 * isolated in the U.S.A. (Iyer and Dobson, 1940).

B1* accidently isolated as a supposed contaminant of an infec-

tious bronchitis virus culture (Hitchner, 1948).

F*, isolated in England in 1949 (Asplin, 1952).

Queensland V4 (Queensland)* isolated in 1966 by Rylie in

Brisbane, Australia (Allan, personal communication).

Ulster*** isolated in Northern Ireland in 1966 from the faeces

of apparently healthy chickens by NcFerran (personal communication).

52

Seed viruses were received from:

*W.H. Allan, Ministry of Agriculture, Fisheries and Food,

Central Veterinary Laboratories, Weybridge, Surrey, England.

**R. Rott, Institute of Virology, Giessen, West Germany.

***J.B. NePerran, Ministry of Agriculture for Ireland, Belfast,

Northern Ireland.

2) Growth of virus

Stock virus and all other virus, i.e. radioactive virus, virus

for treatment with detergent and testing neuraminidase activity,

was grown in eggs.

Fertile hens eggs were obtained from Poyndon Farm Ltd., Goffs

Oak, Hertfordshire, and incubated at 37°C for 9-11 days before use.

Seed virt) or infected allantoic fluid (0.121 of a 10-2 dilu-

tion) was inoculated into the allantoic cavity and the eggs incu-

bated nt 37°C. After 42 hours, with virulent strains, or 72 hours

with avirulent strains, the eggs were chilled at 4°C for several

hours and the allantoic fluid was harvested.

After low speed centrifugation (1000) to remove red blood

cells and any cell debris, virus was either stored as infective ellen-

tole fluid or centrifuged at 40,0001 and resuspended as a more con-

centrated suspension before storing.

Virus suspensions were stored overnight at 4°C or for longer

periods in a -70°C deep freeze.

53

C. CELL CULTURE TECHNIQUES

1) Preparation of chick embryo fibroblast cultures

The heads, legs, guts and viscera of 10-12 day old chick

embryos were removed and the bodies forced through a 10m1 syringe

into sterile PBS. The macerated embryos were washed three times

in PBS and then 100m1 of 0.27w/v trypsin in PBS was added for

every 12 embryos used. The mixture was then stirred at 37°C for

30 minutes after which 10% v/v calf serum was added to inhibit the

trypsin. The suspension obtained was then filtered through a coarse

wire mash strainer and centrifuged for 15 minutes at 1300g. The

supernatant obtained was discarded and the cells resuspended in

growth medium. After passing the cells through a sintered glass

filter they were diluted with 0.17w/v trypan blue in PBS and coun-

ted using a Neubauer counting chamber.

Cells were usually adjusted to 2 x 106/ml for seeding but

sometimes when confluent monolayers were not required concentrations

of 0.6-0.8 x 106 cells/mi were used.

Leighton tube coverslips were seeded with 1.5m1 of 2 x 106/ml,

5cm diameter plastic Petri dishes received 4m1 and 14 cm glass die.-

hes received 2$al to give confluent monolayers in 48 hours.

All cell cultures were incubated at 37°C. Culture vessels

which are not air tight were incubated in an incubator continuously

flushed with a CO2/Air mixture giving a final concentration of 57.

carbon dioxide.

2) Infection of cell monolavers

The required dilution of infected allantoic fluid was made in

maintenance medium and a sufficient volume to cover the surface of

54

the monolayer was used. After adsorption at 37°C for one hour

cells were washed twice with warm PBS or maintenance medium and

fresh maintenance medium added.

The following multiplicities were used:

Experiment Multiplicity

Cell fusion and haemadsorption

10 EID50 /cell

Cell fusion and haenadsorption in

the presence of inhibitors and all

other experiments involving meta-

bolic inhibitors

50 EID50/cell

Double-labelling experiments

50-100 EID50/cell

LDS experiments

50 BID50 /cell

3) Staining call monolayers

a) Giemsa--May GrUnwald

A modification of the method of Jacobson and Webb (1952) was

used. After washing with PBS,coverslip-monolayers to be stained

were fixed in methanol for for 15-30 minutes. The coverslips were

then ibeed in pH6.8 buffer solution containing 5% v/v Giemea solu-

tion and 5% v/m, May GrUnwald solution and left for 20-25 minutes.

The coverslips were then washed once in tap-water and twice in

pH6.8 buffer. After drying under infra red lamps the coverslips

were mounted in DePex (G.T. Gurr Ltd.) for microscopy.

b) Acridine orange

To stain cells with acridine orange the method of Alwyn (1968)

was used.

Michaelis' buffer (Michaelis, 1931) pH4.2 was prepared:

Sodium acetate 9.7g

55

Sodium barbiturate 14.7g

Distilled water to 500m1

For use this buffer was diluted:

Michaelis' Buffer 50m1

Sodium chloride 8.574w/v 20m1

UC1 120m1

Distilled water 60m1

Coverslip cell cultures were immersed in acridine orange 0.017.w/v

In diluted buffer for 10.20 minutes. They were then washed three

times in diluted buffer and mounted in 8%v/v glycerol. Cultures

were then examined under the Leitz microscope using fluorescent light

transmission filter 5mm. B12 and suppression filter K510 or 1530.

In cells stained with acridine orange RUA appeared red while DNA

appeared yellow-green.

Acridine orange solution is extremely susceptible to light and

was therefore stored in a brown glass bottle covered in silver foil

at 4°C.

D. BIOLOGICAL ASSAYS

1) Haemagmlutinin assay

Haemagglutinin (HA) activity vas measured in standard plastic

plates in 0.2m1. volumes. PBS was used as a diluent with 1% chicken

red blood calls.

2) Infectivity

Infectivity for chick embryos was measured by inoculating 0.1m1.

volumes of serially diluted virus suspensions into the allantoic

cavity of 9 or 10-day old fertile chicken eggs. Eggs were candled

daily and after 7 days the allantoic fluid of surviving eggs was

tested for the presence of haemagglutinin. The 50% egg infective

dose (BID50) per ml. was estimated by the Kaerber method.

3) Plaque assay

Primary chick fibroblast monolayers in 5cm diameter plastic

Petri dishes were infected with ten-fold dilutions of virus in PBS

containing 2% foetal calf serum. After adsorption, for one hour, the

inoculum was removed and 4m1 of 0.97.w/v /onager in maintenance media

was added. Dishes were stained with neutral red 0.1%w/v in PBS and

examined for plaques three days after infection.

4) Neuraminidase assay

Neuramimidase (mucopolysaccharide N-acetylneuraminyl hydrolase,

BC.3.2.1.18.) activity was estimated by measuring the amount of N-

acetyl neuraminic acid (NANA) released from a suitable substrate.

The solution to be tested (0.1m1) was incubated with 0.1m1

substrate and 0.3m1 buffer for 10 minutes at 37°C. The NANA rele-

ased was estimated by the method of Aminoff (1961).

The substrate used was either bovine neuramine lactose (vhere,

57

specified) 200pg per test, or the equivalent amount of fetuin.

NANA solutions were used as standards and enzyme activity was

expressed as pg of NANA released.

The buffer was, unless otherwise specified, 0.1H sodium acetate-

acetic acid buffer, pH5.2 for fetuin or pH5.4 for neuramine lactose.

5) Protein assay

The method of Lowry et al (1951) was used for the estimation

of proteins. The final volume was 6.5m1 and optical density (OD) was

read at a wavelength of 600nm. Bovine serum albumin was used as

a standard.

6) Lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was estimated by the

spectrophotometric method described by Kornberg (1955).

7) Estimation of haemadsorption

Coverslip cell cultures were washed three times with ice cold

PBS at the specified times after infection and then received 0.5m1

of a 0.5%v/v suspension of chicken red blood cells. After 15-20

minutes at 4°C, unadsorbed red blood cells were removed by three

further washes with ice-cold PBS. The cultures were then fixed in

methanol, stained with May-GrUnwald.4iemsa, and the number of red

blood cells adsorbed to the cells counted.

The amount of haemadsorption was expressed as the number of

red blood cells adsorbed per cell or per unit area. In experiments

with metabolic inhibitors the number of red blood cells per unit

area in monolayers treated with inhibitor was expressed as a per-

centage of the number in the same area of inhibitor free cultures,

measured 8 hours after infection.

58

8) Estimation of cell fusion

Cell fusion was estimated by one of two methods:

a) Size of syncytia

The size of syncytia WAS estimated from coverslip monolayers

of chick embryo cells stained with Giensa-May GrUnwald 12 hours

after infection. Size was expressed as the average number of

nuclei seen in syncytia at that time.

b) 7. polykaryocytosis

The extent of cell fusion was estimated from confluent cover-

slip monolayers stained with Giemsa-May Grunwald 15 hours after

infection. The results are expressed as the number of nuclei invol-

ved in syncytia in a given area, i.e. one field of the microscope,

as a percentage of the total number of nuclei in that area.

In experiments with metabolic inhibitors the % polykaryocy-

tosis with treated cells is expressed as 7. of the polykaryocytosis

in inhibitor free infected-cultures

9) Estimation of call-associated neuraminidase activity

For each strain tested 1 x 109 EID50 were inoculated into the

allantoic cavity of 10-day-old eggs. After 22 hours incubation

at 37°C the chorioallantoic membrane was removed from each egg,

washed three times in ice-cold PBS and stored at -70°C until assayed.

The membranes were then thawed, and blended in homogenizers with

motor driven Teflon pestles in 0.0114 sodium acetate-acetic acid

buffer, 05.2. The homogenate was then assayed for neuraminidase

activity and protein.

J r

B. VIRUS DISRUPTION

1) Sodium dodecyl sulphate (SDS) 2-marcaptoethanol

Virus disruption prior to acrylamide gel electrophoresis

was done by suspending pelleted virus in 17. w/v sodium dodecyl

sulphate (SDS) and 1% w/v mercaptoethanol (Shapiro, Vinuela and

Maisel, 1967) or by making suspensions this concentration by addi-

tion of 107. w/v SDS-mercaptoethanol. The mixture clarified immed-

iately but was heated for 30-60 minutes at 37°C. No difference was

seen in the SDS-gel pattern of disrupted virus if 17. w/v dithio-

threitol (DTT) was used instead of mercaptoethanol (Gordon, 1969),

the mixture was heated at 100°C for 1 minute - (Maisel, White and

Scharff, 1968) or if 0.5M urea was present.

When run in parallel with double-labelled cell extract, i.e.

at the same time but not in the same tubes, virus was disrupted by

the method of Summers, Maizell and Darnell (1965, also sae below),

however no difference in gel patterns was seen when compared to ord-

inary SDS disrupted virus gels.

2) Tween 20 at pH10

The method used to disrupt virus with Tween 20 was that of

Webster and Darlington (1969). Virus was suspended 17v/v Tween 20

in 0.02M bicarbonate buffer, 010, The mixture was held at 37°C

overnight and intact virus removed by centrifugation at 45,000g for

30 minutes.

Tween 20 was separated from the supernatant and partial separa-

tion of neuraminidase and HA activities was obtained by separation

on a 0200 sophadex column.. A column of Sephadex G200 was made in a

60

K25/100 Sephadex glass column (Pharmacia, Uppsala, Sweden). The

length of the column was 35cm and the width 2.5cm. The samples were

applied to the bottom and elution with PBS was upwards using an

LKB peristaltic pump run at 10ml/hr., 3 ml. fractions were collected.

The column was kept at 4°C.

3) Sodium deoxycholate

To disrupt virus with sodium deoxycholate a modification of

the method of Kingsbury and Darlington (1966) was used. Virus was

pelleted by centrifugation at 40,0008 for 40 minutes and resuspen-

ded to a concentration of 1mg/m1 of protein in 0.01M deoxycholate.

RNP was separated from the disrupted virus membrane by sucrose den-

sity gradient centrifugation. The gradient was made 5-407.w/v

sucrose in 0.01M DOC and centrifugation was in a 3 x 23m1 swingout

rotor at 4°C for IA hours at 60,000g.

61

F. RADIOACTIVE ISOTOPE COUNTING TECHNIQUES

1) Scintillation counter

The counter used to count all labelled materials vas an ABAC

S1.40 liquid scintillation spectrometer: Intertechnique Ltd.,

Portslade, England.

2) Scintillation fluids

a) Non aqueous samples

Non aqueous samples, including dried coverslip monolayers and

dried glassfibre filters, were counted in 15 or 10m1 of scintillation

fluid prepared:

Butyl-PBD (Intertechnique Ltd.) 8.0g

Toluene (Scintillation grade) to 1000m1

b) Aqueous samples, including dissolved polyacrylamide gel

fractions were counted in 15m1 of scintillation fluid prepared:

Butyl-PBD. 8.0g

Naphthelene (Intertechnique Ltd.) 80.0g

Thixotropic gal (Cab-O-Sil, Packard Instruments Ltd.,

Wembley, London) 40.0g

Made up to 1000m1 with a 2-ethoxyethanol 40%v/v:

Toluene 60%v/v mixture.

Cab-O-Sil is used to keep any precipitate that may form evenly

distributed throughout the scintillation fluid (Ott et al, 1959).

3) Estimation of uptake of radioactive isotope into coverslip cell

cultures

To estimate the amount of uptake of radioactive labelled pre-

cursor into macromolecules in coverslip cell cultures the following

62

method was employed.

After removal of the media containing the radioactive precur-

sor cultures were washed twice in ice cold PBS and three times in

ice cold 5Zv/v trichloracetic acid (TCA). After a further wash in

acetone the coverslips were air-dried and then transferred to 15m1

toluene butyl PBD scintillation fluid for estimation of radioactivity.

Maintenance media containing 1.0µC/ml of 311.4eucine was used

to pulse label coverslip call cultures for 30 or 60 minutes in

experiments involving protein synthesis.

In RNA-uptake experiments 1.0µC/ml in maintenance media vas

used, in the presence of 5µg/m1 Actinomycin D but accumulation of

counts was measured.

4) Estimation of radioactivity_ in Bel slices

Polyacrylamide gels containing radioactive labelled polypep-

tides were fractionated using a Mickle gel slicer (Joyceuloebl Ltd.,

London, England) into slices 1mm thick. Each slice vas then diss-

olved in "100 volumes" hydrogen peroxide by incubating for 1-2

hours at 90°C or 60°C overnight (Young and Fulhorst, 1965). This

treatment also decolorises gels previously stained with amid*

black. Scintillation fluid (15m1, of 2 -Othoxyethanol-toluane4ab-

0-sil) was then added to the dissolved gel and the radioactivity

counted. Usually the vials were held at 4°C for a few hours before

counting as this facilitated the removal of air bubbles by gentle

shaking.

5) Quench curve

The estimation of radioactivity due to 14C and 311 in a double

labelled sample using liquid scintillation spectrometry poses sav-

63

eral problems. The energy spectra of disintegration of 14C and 3H

overlap in the machine so that without some method of resolution it

is not possible to distinguish the CPM contributed by each isotope.

Furthe4 many substances, in this case dissolved acrylamide gel,

reduce the counting efficiency of, or 'quench' these isotopes

causing an increase in the overlapping of the spectra. The degree

of quench relates to the concentration of the quenching material.

To separate 14C counts from 3H counts in a double labelled

sample a quench curve (i.e. the relationship between efficiency of

counting and the amount of quench) was constructed. This was done

using the external standard ratio method described in the Intertech-

nique Handbook for the ADAC SL4O spectrometer which approximates

the counting efficiency/quench relationship to a third order poly-

nomial curve. This enables the machine to compute the cross-

contribution of 14C activity to the 3H spectrum and calculate the

disintegrations per minute of each sample.

Standard radioactive solutions were made using 14C-hexadecane

and 3H-hexadecane. Quench material was polyacrylamide gel dissolved

in hydrogen peroxide.

Occasionally dissolved fractions of gel formed a fine precipi-

tate on addition of scintillation fluid. However because Cab-06-

sil keeps fine precipitates evenly distributed throughout the

scintillation fluid the quench curve could be constructed using

the 'external standard ratio' method.

The curve constructed gave correct disintegrations per minute

(DPM) of double labelled standards to within ±1% of the known

radioactivity.

64

6) Radioactive virus

Radioactive virus was grown in an identical manner to unlabelled

virus except that prior to Infection radioactive precursor was

inoculated into each egg. Usually 5pC 14C-labelled precursor or

10pC 38-labelled precursor were added in 0.1m1 of PBS to each egg.

65

G. POLYACRYLAHIDE GEL DISCELECTROPHORES/8

The method used is basically a modification of that of Ornstein

(1964) and Davis (1964).

The apparatus used was the standard 'Shandon' equipment

(Shandon Scientific Co. Ltd., London,England) except that glass

running tubes were either: 0.7cm internal diameter, 8.5cm length;

or 0.5cm internal diameter, 7.5cm length.

1) Preparation of 7.5% eels

The following stock solutions were made and stored at 4°C.

a) Buffer

Tris (hydroxymethyl) methylamine (Tris) 36.6g

N,N,N ,N1:Tetramethylethylenediamine (TED) 0.23ma

IN Hydrochloric acid to p88.9 approximately 48ml


b) Acrylamids

30g

Distilled water to

100m1

c) N,Hr-methylenebisecrylamide (BIS)

cP5g


To prepare 7.3% acrylamide gels the above solutions were mixed:

a) one part,

b) two parts

c) one part

Distilled water four parts

The mixture was deearated by vacuum and made ' 0.6%v/v with ammon-

ium persulphate solution (7%w/v), shaken gently in a circular motion

so that reiteration did not occur. Glass tubes, suitably stoppered

with rubber or plastic caps, were then filled with mixture to lcm

from the top of the tube. A distilled water overlay was then

66

added with sufficient care to avoid disturbing the interphase. A

syringe was used and the needle pressed against the side of the

tube to avoid such mixing. The water overlay serves two purposes;

it excludes air, which prevents polymerisation, and produces a flat

interphase. The gels were then allowed to polymerize, this took

10-20 minutes at room temperature.

2) Running

Protein samples to be run were prepared in the same buffer as

the reservoir buffer but at 1/10 concentration, which helped sharpen

the bands. Samples were made 10%v/v with 60%w/w sucrose and 107.

v/v with 0.001%w/v Bromophenol blue or 0.001%w/v Phenol red and

layered carefully onto the surface of the gels through the reservoir

buffer with a 'Hamilton' syringe.

A current of 1mA per tube was applied for 15 minutes followed

by a current of 4mA per tube until the marker had migrated a pre-

determined distance, usually 4.5cm. Migration was from the cathode

(-) to the anode(+), i.e. top to bottom in this case.

The reservoir buffer in all the experiments described was

made:

Tris 10.0g

Disodiumethylendiaminetetra-acetic acid (EDTA) 1.0g

adjusted to pH9.2 with saturated boric acid solution and made up

to 1000m1 with distilled water. In experiments in which proteins

treated with SDS were run the reservoir buffer contained 0.17w/v

SDS.

3) Staining

Gels were removed from the tubes by rleming with a needle while

7%w/v acetic acid was injected between the gel and the glass. They

67

were then fixed and stained overnight with 1%w/v amido black in 7%

acetic acid. Excess stain was removed in one of three ways:

a) Repeated washing and soaking in 7% acetic acid.

b) Repeated washing and soaking in 20%v/v ethanol in

7% w/v acetic acid at 60°C.

c) Transverse destaining using a "Transverse Disc Destainer"

(Shandon Ltd.).

Unstained gels for densitometer scanning were fixed in 5%w/v

TCA and stored at 4°C.

4) Estimation of nolypentide molecular veizht

The method of Shapiro, Vinuela and Maizel (1967) vas used,

except that 7.5% acrylamide gals were used.

Marker proteins were all obtained as "Non enzymic protein

molecular weight markers" from Hann Research Laboratories Inc., New

York, U.S.A.

68

H. DOUBLE LABELLING TECHNIQUE

The method described below is a modification of the methods of

Summers, Maizel and Darnell (1965) and Hay, Skehel and Burke (1968).

Chick embryo cell monolayers were grown in 14cm glass Petri

dishes and half the total dishes were infected with 50-100 E/D50/

cell of the strain under investigation. After adsorption for one

hour the cells were washed twice with PBS at 37°C and 20m1 main-

tenance media containing actinomycin D was added. Actinomycin D

was used at a concentration of 5pg/ml for samples up to the sixth

hour and 1pg/ml for later samples. At the times specified cells

were washed twice with warm Earle's saline (Earle 1959) containing

2%v/v foetal calf serum and the following amount of radioactive

amino acid added:

a) To infected cells,

10m1 of Earle's saline + 27 foetal calf serum, containing

10µC/ml 3H-leucine.

b) To uninfected calls,

10m1 of Earle's saline + 2% foetal calf serum, containing

_ -. 2.5pC/m1 14 u Leucine.

After a one hour pulse, cultures were washed twice with ice cold

PBS and then once with ice cold hypotonic buffer (magnesium chloride

103M, Tris/HC1 10-3M pH8.3, 2-mercaptoethanol (0.1%v/v) 1.4 x

10-214). The cells were scraped from the Petri dishes into one ml

of hypotonic buffer and the infected 3H-leucine labelled cells

mixed with the uninfected 14C-leucine labelled cells.

The cell mixture was made 107.v/v with glacial acetic acid,

69

0.5M with urea and 17.w/v with SDS. The mixture was then incubated

at 37°C for 30-60 minutes and dialysed overnight against 2000-3000

volumes of 1/10 TEB pR 9.2, 0.17. SDS, 0.1% 2-mercaptosthanol and

0.5M urea, at room temperature.

The dialysate was decanted from any insoluble material remain-

ing and adjusted to 2mg protein per ml. Samples of 0.1m1 were then

analysed by electrophoresis on 7.57. acrylamide gels. At the end

of a run the gels were stained in amido black and then sliced, diss-

olved in hydrogen peroxide and radioactivity estimated. The radio-

activity was expressed as a ratio of 311 DEM to 14C DPM. Because

4 times the concentration of 3H-leucine compared to 14C-leucine was

used ratios were divided by 4.

70

J. DENSITY GRADIENT CENTRIFUGATION - VIRUS PURIFICATION

Preliminary purification and concentration of virus was done

as described above. Depending on the quantity of virus to be puri-

fied one of the following rotors for the MSE 65 centrifuge was

used for further purification on density gradients: B XIV zonal

rotor, D XV zonal rotor, 3 x 65m1 swing out, 3 x 23m1 swing out and

3 x 10m1 swing out.

Linear sucrose or tartrate gradients were prepared using the

relevant size MSE gradient mixer (M.S.E. Ltd., London, England).

Tubes were pierced using an MSE tube piercer and all fractions

collected on the LKB ultroi-4c fraction collector. Ultraviolet

adsorption was measured on the LKB uvicord.

Sucrose and potassium tartrate solutions in 0.01M Tris/HC1

buffer pH7.4 wore made as w/w solutions in accordance with the

Handbook of Chemistry and Physics (Weast, 1968).

The refractive index of solutions was measured using a refrac-

tometer (Hilger Watts Ltd., London, England) at 20°C. Densities

were calculated from the refractive index and are expressed as g/ml

at 20°C.

Radioactivity in fractions after density centrifugation of virus

containing 3H-uridine was measured after precipitation on to glass

fibre filters with ice cold 5% TCA, washing each filter twice with

ice cold TCA and drying the filters under infra red lamps before

transferring them to scintillation fluid for counting.

71

/II RESULTS

A. VIRUS PURIFICATION AND BIOPHYSICAL PROPERTIES

1) Linear sucrose gradient

A typical result of centrifuging strain Italian on a linear

sucrose gradient using a B XIV zonal rotor is shown in Figure 3.

There are two major protein peaks, one is found at the top of the

gradient while the other is associated with the major haamagglu-

tinin peak at a density of 1.19g/ml. Another minor peak of HA

activity can be seen at a density of 1.12-1.13g/m1 while some HA

remains on top of the gradient.

2) Linear tartrate gradient

The separation of virus (strain Italian) on a linear potassium

tartrate gradient using a BXIV zonal rotor is shown in Figure 4.

The over all appearance is similar to the results on a linear sucr-

ose gradient, however, the major HA peak appears at a density of

1.22g/m1 while the minor HA peak is found at 1.16g/ml.

3) Association of viral prozerties to peaks

Virus (strain Italian) labelled with 3H-uridine was prepared

and run on a linear sucrose gradient in a 3 x 23m1 swing out rotor.

Figure 5 shows that an acid-insoluble radioactivity peak and infec-

tivity are identifiable with the major HA peak at a density of 1.19

g/ml. The minor HA peak shows little infectivity and appears to

possess no RNA. These results confirm that the whole virus is

found at a density of 1.190m1 in sucrose. Similar experiments

showed that the peak at 1.22g/m1 in potassium tartrate is mature virus.

Electron microscopy confirmed these finding* typical intact virus

72

Ha

em

ag

glu

tin

sn

pe

r m

l x

10

00

M.

M:

M.

M-

12."

2.1

Euo 01

10-

7

Z" '"11111,1111 muumuui

•

FIGURE 3

73

.1 06

4 e 12 16 20 24 2. 32 36 40 44 45 12 66 60 64

Fraction number (10 ml each)

Purification of strain Italian on a linear sucrose gradient;

BXIV rotor, centrifugation: 40,000rpm for 90min.

Histogram is the reciprocal of HA titre.

• solution density.

protein.

.. r .

200

HA

100

C a)

12 .w•. ♦......

..... • ............ • ........... A

............ • .........

.....

•

40"•••••%/4/

.........

FIGURE 4

74

30

40

Fraction (10m1)

Purification of strain Italian on a linear potassium tartrate

gradient; OXIV rotor, centrifugation: 45,000rpm for 16hr.

Histogram is the reciprocal of the HA titre.

,••

0 c) I-x

E a-C.)

- s

- a

-4

20-'

18-

18-, o o -

E 12- - 10

1.24 .-.

3

2 4 8 8 10 12

FIGURE 5

Fraction number

Purification of strain Italian on a linear sucrose gradient;

3 X 23m1. awing-out rotor, centrifugation: 30,000rpm for 90min.

o solution density.

• infectivity. _

• CPM due to 3H-uridine uptake into TCA-insoluble material.

75

could be seen at the higher density while the minor HA peak revealed

what appeared to be fragments of virus or modified cell membrane.

4) Discontinuous gradients

Experiments were carried out to investigate the possibility

of using the simpler technique of discontinuous gradients. Succ-

essful separation of virus was obtained using gradients made from

sucrose solutions of the following densities: 1.04, 1.16, 1.26.

and 1.288/m1 in the ratio of 1:2:1:eto fill'. These gradients

possess two 'steps' in which the density changes rapidly (Figure

6) because of this the virus peak is much sharper and appears in

fewer fractions. If centrifugation was continued for long periods,

i.e. 12-18 hours or discontinuous gradients were left for several

hours before use, mixing of the bands occurred and shallower steps

in the gradient were obtained. With potassium tartrate solutions

discontinuous gradients could not be obtained: evidently the lack

of viscosity of tartrate permitted wady mixing so that almost linear

gradients were formed.

5) Recentrifugation of HA peaks

The occurrence of two haemagglutinin peaks, one of which was

possibly virus fragments could be caused by degradation of the virus

during the purification procedure. To discover if this was so

material from fractions corresponding to the two major HA peaks on

a sucrose gradient was sedimented and recentrifuged on sucrose

gradients. The single peak obtained from the whole virus at density

1.19g/m1 (Figure 7) suggests that the procedure does not degrade

intact virions. Recentrifugation of the lower density HA peak

showed that some whole virus is also associated with the fragments

(Figure 8).

76

1.22

1.20

1.18

E 1.10

0

1•10

munc 6

77

40

0 0 0

30

20

5

0 0

10

am ro 10,0,,000644//0/0/ Cd7 ALW71,

4 8 12 18 20 24 28 32 38 40 44 48

Fraction number (icimi each)

X

Purification of strain Italian on a discontinuouo sucroao

gradient; 3XIV rotor, Contrifugations 40,00Orpm for DOmin.

Histogram is tho reciprocal of the HA titre.

1 20

E ......

CO

118

1.10

1 22

1.10 H

aem

agg

luti

nin

per

ml x

10

00

4 8 12 18 20 24 28 32 38 40

FIGURE 7

78

Fraction number (to ml each)

Recentrifugation or major HA peak obtained from a gradient

similar to that seen in Fig. 3. EIXIV rotor, sucrose gradient

centrifugation: 40,000rpm for 90min.

FIGURE 8

79

5 10 15 20

Fraction

Rocentrifugotion of minor HA peak obtained from a gradient

similar to that seen in Fig. 3. 3 X 23m1. ewing*out rotor.

centrifugations 35od0Orpm for 180min., sucrose gradient.

I 30

E I 20

); 1 C 0 0

110

1

- 15

- 10 N

9

X

< I

- 5

6) Nature of major and minor HA peaks

Lack of RNA and infectivity, together with electron microscopic

evidence all suggest that the minor HA peek found at a density of

1.13 when strain Italian is run on a sucrose gradient consists of

virus fragments or modified cell membrane fragments. When whole

NDV is disrupted with SDS polyscrylemide gel discelectrophoresis

yields 3 major bands (see below). When samples from the major HA

peak and minor HA peak were treated in this manner, the major HA

peak showed 3 major bands while the minor HA peak showed only 2

bands the middle band being absent (Figure 9).

7) Separation undpr varying conditions

Whole virus banded at its isopycnic density after very short

runs, as brief as 45 minutes at 45,000 r.p.m., or long runs, 18

hours at 45,000 r.p.m., on linear or discontinuous gradients (Table

4). This is an important point, because virus is so easily sedi-

mented to its isopycnic density short runs are preferable for virus

purification since under these conditions virus is subjected to

both isopycnic and rate zonal separation. Cell fragments which may

have the same density as virus but a slower sedimentation rate are

prevented from contaminating the virus. This'is less desirable if

contaminating proteins of greater density than the virus are present,

but results have shown that these are minimal. Where protein peaks

have appeared at densities greater than the virus peak electron

microscopy has shown them to consist of RNP fragments.

8) Estimation of virus purity

Estimation of purity poses a considerable problem. Purifica-

tion can be monitored by measuring HA per mg protein at each step

(Table 5). By this method it can be seen that virus after zonal

80

81

A B

g in HA

nt.

d H p k • 1 nd '. di ru t d 1 1 SO -

d

b •

- H

H p •

TABLE 4

Densities at which haemagglutinin peaks occurred on sucrose gradients

under different conditions. Virus: Italien.

Length of run R.P.M. Density Type of Gradient (Hours) (BXIV rotor) ( g/ml at 20°C)

HA Peaks

I II

45,000 1.19 1.13 Discontinuous

1. 45,000 1.19 1.13 Linear

11/2 45,000 1.18 1.14 Discontinuous

2 45,000 1.185 1.13 Discontinuous

3 47,000 1.18 1.12 Linear

14 30,000 1.19 1.12 Linear '

18 30,000 1.195 1.13 Linear

15 47,000 1.18 1.13 Discontinuous

82

TABLE 5

Purification of NDV strain Italian

83

HA/ml Protein ,ug/m1

HA per mg protein

5,120 2,000 2,560

163,840 2,600 63,000

40,960 50 819,200

Sample

Original allantoic fluid

Concentrate from allantoic fluid

Virus after zonal centrifugation

centrifugation is 320-fold purer than infected allantoic fluid;

however this does not show that no contaminating proteins are pres-

ent. Purification can also be monitored by the use of polyacryla..

mide gel discelectrophoresis. Whole virus will not enter 7.5%

acrylamide gels while smaller soluble proteins will, figure 10

shows therefore that after sonal centrifugation virus is free of

soluble proteins. Bands of 1 -2pg of cytochrmhs C can be seen quite

distinctly on similar gels. Soluble proteins too large to enter

the gel could be present but this is unlikely since purified virus

passed through both a sephadex G200 column and a DEAE DE32 (Whatman

Ltd) column as a single protein peak.

9) Behaviour of different strains of NDV on density gradients

The buoyant densities of the whole virus and the minor HA peak

of seven different strains of NDV which differ in virulence was

measured on gradients of sucrose, potassium tartrate and caesium

chloride. Very little variation was seen amongst the whole virus

of the different strains on sucrose, the range was 1.19-1.20g/m1

and caesium chloride, the range was 1.20-1.21; potassium tartrate

gradients gave more heterogenous results the range was 1.20-1•.22

g/ml but this was not considered significant as there is no appar-

ent trend if compared with virulence (Table 6). The high density

of virus on potassium tartrate and caesium chloride gradients is

presumed due to the uptake of these low molecular weight salts into

the virion while this does not occur with sucrose.

The minor HA peak was more heterogamous, having a range of 1.13-

1.16g/ml on sucrose and 1.12-1.168/m1 on tartrate. These ranges are

no greater than have been recorded in different preparations of any

one strain and should be expected if this band is fragments of virus

84

85

(1

411

Purification of NtV. The fallowing were diluted so that

0.1m1. layered on to polyacrylemide gels contained 2094 of

proteinta) Original infected allantoic fluid. b) Allantoic

fluid after low speed centrifugation. c) Pellet obtained after

high speed centrifugation. d) Supernatant obtained after high

speed centrifugation. a) Virus suspension after zonal cent-

.rifugation.

86 TABLE 6

The buoyant densities of the major and minor haemagglutinin peaks of different strains of NDV.

Strain Density g/ml at 20°C in:

Sucrose

I II

Sodium tartrate Caesium chloride

Harts (BHK) 1.19 1.15 1.21 1.12 ND

Herts 1.19 1.14 1.20 1.14 1.20

Italien 1.19 1.13 1.22 1.16 1.21

Texas ND ND 1.20 ND 1.20

Beaudette C 1.19 1.16 1.22 1.16 1.21

B1 1.20 1.15 1.215 1.15 1.21

Queensland 1.20 1.16 1.21 1.14 1.21

Ulster 1.19 1.14 1.20 1.13 ND

Herts (BHK) was strain Herts grown in suspended BHK cells.

All other strains were grown in eggs.

ND Not determined.

I vs Whole virus peak.

II ==, Minor HA peak.

or modified cell membranes. There is little difference between

the lamyant density of the minor peak in sucrose and tartrate as

there is not complete membrane present. In caesium chloride grad-

ient experiments already purified virus was used.

10) Discussion of results

Satisfactory separation of NDV from large volumes of infected

allantoic fluid vas obtained using either sucrose or potassium

tartrate gradients in zonal rotors. Discontinuous sucrose gradients

simplified the technique without affecting the purification.

Separation of smaller volumes of virus on gradients made in

tubes in Ewing out rotors was less successful. However using the

following procedure pure virus could be readily obtained: 1) After

concentration virus was centrifuged through potassium tartrate sol-

ution at a density of 1.16g/m1 onto a sucrose cushion at a density

of 1.24 using a Sorvall centrifuge at 40,000A for 2 hours. 2) The

band at the interphase was collected and after dilution was placed

on a linear gradient of 0-60% sucrose and spun at 22,000 r.p.m. in

the 3 x 65m1 rotor for 2 hours. In this way satisfactorily puri-

fied virus could be obtained.

Since the initial experiments gradients of potassium tartrate

were seldom used. Generally, purified virus was needed for treat-

ment with SDS and the presence of potassium causes precipitation

of this detergent.

From the results it can be seen that virulent strains do not

differ significantly in bubyant density from avirulent strains.

Stonback and Durand (1963) showed that the density of the virion

was host controlled and some variation might have been expected as

87

Bang (1953) reported that avirulent and virulent strains differ

in their site of maturity within the cell. However if such diff-

erences exist they may not be large enough. to detect using these

techniques. In this respect NDV is unlike the herpes simplex virus

system in which it has been shown that strains differing in viru-

lence differ in buoyant density (Holzman and Roane, 1961).

88

B. THE STRUCTURAL PROTEINS OF NEWCASTLE DISEASE VIRUS

1) Polyacrylamide gel discelectrophoresis after disruption by SDS

Discelectrophoresis on 7.5% polyacrylamide gels in the presence

of 0.1% SDS after disruption of purified virus with 1% SDS and 17

mercaptoethanol reveals three major polypeptide bands, I, II, III

and one minor band, A, after staining with amidoblack (Fig. 11,

photograph). Occasionally other minor bands were seen B, C, and

IIa (Fig. 11, diagram).

2) Polypeptide patterns of different strains

The polypeptide patterns seen with all the strains tested were

essentially identical. Three major and one minor bands were seen

consistently. The migrations of these bands as percent migration

of bromophenol blue or phenol red markers are shown in Table 7.

The values for Beaudette C (a) and Beaudette C (b) are the extremes

recorded with this strain, while the migration of the bands of

the other strains is an average of at least three gels. It can

be seen that the variation from strain to strain is no greater

than may be recorded using the same strain.

3) Radioactive labelled virus

Labelling virus with radioactive amino acids confirms the

peptide nature of the bands seen in stained gels, since radioactivity

appeared'in peaks corresponding by migration to the stained bands,

Figures 12, 13 and 14 show that all four strains tested have the

three major bands seen in stained gels and although the minor band

A is hardly above background it may be distinguished as a minor peak.

Figure 12 shows analysis of two strains, Texas and Beaudette C,

labelled with 14C and 3H-leucine respectively, after electrophoresis

on the same gel, it can be seen that all peaks have exactly tha came

migrations.

89

FIGURE 11

90

A

B

C

ila

Bromophenol blue

NA structural proteins.

The photograph of the gel shows typical separation of NA

polypeptides (Herts) after polyacrylamide gel electrophoresis

in the presence of 505 after TOS-mercaptoethanol disruption.

Staining: Amido black.

The diagram shows thu minor bands occasionally seen and

their migration in relation to the major polypeptide bands.

..0,011.1710P4,Trwt.c. Origin

TABLE 7

Migration of the polypeptide bands of different strains of NDV

on 7.57 polyacrylamide gels.

Strain Band

Distance migrated 7. bromophenol blue

Herts 8.5 45.0 59.0 70.0

Texas 8.5 48.0 62.0 72.0

*Beaudette C (a) 8.7 50.0 63.0 73.0

*Beaudette C (b) 9.5 45.0 60.0 69.0

B1 9.5 45.0 60.0 69.0

Queensland 9.3 46.5 61.0 70.0

Distance migrated as 7. phenol red

Warwick 16.5 53.0 69.0 73.0

Beaudette C 15.0 50.5 69.5 78.0

F 14.5 51.0 70.0 76.5

Queensland 10.5 48.0 66.0 76.0

Ulster 13.5 53.0 69.0 76.0

* Extremes recorded for this strain. The other fesults are the average

of at least three runs.

91

3

141C -DPM x 10-2

2

11 111 A

Beaudette C 3H- !suckle

Texas 14

C-leucine

12

10 3H-DPM

x102

..._ ..a...

10 20 30

Fraction Number 40

FIGURE 12

92

Separation of SOS.mereaptoethanol disrupted purified virus

by polyacrylemida gal electrophoreois. 3H6pleucine labelled

Beaudette C (v) and 14C.leucine labelled Texas ( • ) ware

run on the same gel. Fractions represent 1mm nlicss of gel.

FIGURE 13

93

8 I A II

land

7

6 —

5 —

N 4 oc,

2 3 a.

2 X

0

0

450

100

80 IL C.)

60

40

20

1-1 10 20 30 Fraction Number

40 (+)

Saparation of LDS-morcaptoothanol disrupted virus by poly-

acrylamide gel electrophoraais. Strain Queensland, 3H-loucine

labelled (o) and 14C-glucosamina libelled ( v ) run on separate

gels.

FIGURE 14

94

1

1

1

Herts 3H- arginine

200

DPM

100

(-) 10 20

30

40

1+)

Fraction Number

Savration of 0.5-mercvtosthanol disrupted purified virus

by pelyecrylamicio gel olectrehoresis. 3H-arginine inbollnd

Herts.

4) Relative proportions of the polypeptides

The proportion of the total protein found in any one band can be

estimated either by measuring the amount of radioactivity in any

band after labelling or by measuring the area under the curves of

a stained or unstained densitometer trace of 600 nm or 280 nm

wavelength respectively. Within the limitations of the method

the results are remarkably consistent for all the methods of

estimation used (Table 8). The average values are: I - 21%,

II • 44%, III - 307., and A • 2.5%. The greatest variation was with

polypeptide II which ranged from 40.57. (Harts stained trace) to 46.07.

(Queensland, 3H-leucine), however the figures of, 44.67. for Texas

and 41.6% for Beaudette C suggest that this is more likely to

be an experimental variation than a real difference and is certainly

not related to virulence. In comparison with the results of

Haslam et al (1969) polypeptide III shows a much higher proportion,

307. compared to 217.,while the others are similar.

Table 8 also shows the molecular ratios of the polypeptides,

these indicate the relative ratio of the molecules of each poly-

peptides in the virion. The strains tested are remarkably similar

in:this respect. Labelling with 3H-arginine indicates that

polypeptide I is particularly low in this amino acid.

Virus (strain Queensland) was also grown in the presence of

radioactive glucosamine as this specifically labels the glycoproteina

(Klenk, Caliguri and Choppin, 1970; Burge and Strauss, 1970).

Although counts were low due to the low specific activity of the

available isotope the results show that there is a peak of activity

at the position of polypeptide I which indicates that this is a

glycoprotein (Fig. 13). Similarly there is a somewhat smaller

peak at polypeptide II which suggests that this is also a glycoprotein,

while although there is a small peak at the position of polypeptide

III it seems unlikely that this is of sufficient significance to

demonstrate that polypeptide III is a glycoprotein.

95

96 TABLE 8

Relative proportions of NDV polypeptidea

Strain 7. Total yrotein !lethod

Molecular ratio*

III A Rest

V-8 VB1/66 18 45 21 5 11 Haslam et al

(avirulent) '0.42 1.54 1.0 (1969)

Texas 21.0 44.6 30.5 2.7 1.2 14C-leucine 0.33 1.06 1.0 0.019 -

Beaudette C 21.2 41.6 31.0 2.8 3.5 3H-leucine

0.32 0.97 1.0 0.018 -

Queensland 19.1 46.0 29.7 2.5 2.7 3H-leucine

0.31 1.12 1.0 0.018 -

Herts 21.0 40.5 30.6 7.9 Amido black

0.33 0.96 1.0 stained trace

Herts 21.6 40.5 32.9 5.2 Unstained trace

0.31 0.90 1.0

Herts 12.0 45.0 31.0 12,0 3H-arginine

0.18 1.05 1.0

k The molecular ratios were calculated by dividing the percenta3e of virus

protein by the molecular weight of the polypeptide. The resultant ratios

were then divided by the value for peak III so that the results may be

compared.

97 5) Estimation of molecular weight of the polypeptides

Treatment of proteins with SDS produces a rod-like particle the

length of which varies with the molecular weight of the protein in the

complex (Reynold and Tanford, 1970). Because of this property tha

distance of protein migration on polyacrylamide gels in the presence of

SDS is a direct consequence of the molecular weight of the protein. In

fact migration shows a straight line relationship to the log of the

molecular weight (Shapiro, Vifluela and Maizel, 1967).

This method has been used to estimate the molecular weights of the

NDV polypeptides (Fig. 15). The results obtained are shown in Table 9

together with the molecular weights reported by other authors using a

similar technique. The range from experiment to experiment is considerable -

84-90,000 for pblypeptide I, 56-60,000 for II, 40-42,000 for III - however

the figures obtained are a useful approximation. The average molecular

weight for each polypeptide compares favoUrablY' with the average of the

previously reported values although these have even greater ranges.

Polypeptides in the region of polypeptide A have a large molecular

weight of 200,000 or more which suggests that this may be due to incomplete

dissociation of virus or reeggregation of polypeptides. The occasional

bands B, 160,000 and C, 120-140,000 may be produced in a similar way. The

other occasional band, tIe, which has a molecular weight of 50,000 appears

to correspond to the polypeptide in this position obtained from

nucleocapsid isolated after tryptic digestion of infected cells

(Mountcastle et al 1970).

6) Identification of NDV polypeptides

a) Nucleocapsid polypeptide.

Treatment of pure virus, strain Harts, with deoxycholate disrupts the

virus membrane and releases RNP. RNP can then be separated from the

disrupted membrane by rate zonal centrifugation on a sucrose gradient

Queensland

15 2x BSA

MW 10

X10 4 8

2 x0VALBUMIN

0 IH.L )

BSA

4

6 HO

1 \ \\Sf\

ii OVALBUMIN

ill PEPSIN

2

FIGURE 15

98

30 40 50 60

70

80

Migration 7. Bromophenol blue

Estimation of the molecular weights of NOV polypeptidas

by polyscrylamide gel electrophoresis. 5DS-merceptoethanol

disrupted virus (e.g. Queensland) was run in parallel with

the following marker proteins of known molecular weight*

H or L = heavy or light chain ofp.globulin (human); OSA m

bovine serum albumin; ovalbumin; pepsin. Each gel wee run

with a bromophenol blue marker.

99

TABLE 9

The molecular weights of NDV polypeptides

Strain

Polypeptide Source

I II III

M.W. X103

Beaudette C 100 62 45 Bikel cC Duesberg

(1969)

Beaudette C 90 62 42 Evans S. Kingsbury

(1969)

V-8 VRI/66 80 54 38 Haslam et al (1970)

Herta 85 40 58

Herts 84 56 42'

Herts 95 60 42

Beaudette C 90 57 42

Oueensland 88 57 42

AVERAGE 88 58 42

100 containing deoxycholate (Kingsbury and Darlington 1968). Figure 16

shows the separation of RNP on a sucrose gradient.

The RNP obtained was further purified by separation on a caesium

chloride gradient, banding at a density of 1.30 g/ml. After dialysis

against PBS and centrifugation the resuspended pellet was shown to

consist of RNP by its characteristic spectrum from wavelength 240 nm

to 320 nm (Fig. 17) which is identical to that shown by Kingsbury

and Darlington (1968).

Treatment of the purified RNP with SDS separates the nucleocapaid

polypeptide from the RNA. Analysis of SDS-treated isolated RNP by

polyacrylamide gel electrophoresis showed a single polypeptide which

corresponded to polypeptide II of the whole virus, having a migration of

59.57. of that of bromophenol blue compared to 60.67. of the migration of

polypeptide II of whole virus (Table 10).

b) Neuraminidase and haemagglutinin

It has been shown that treatment of NDV with TWeen 20 at pH10

releases the HA and neuraminidase without destroying their activities,

although HA activity is prevented in the presence of A'ween 20 (Vebster

- • •-•:-• end Darlington 1969).

The supernatant obtained after centrifugation of purified virus

(Ulster) treated with Teen 20, was applied to a 0200-sephadex column

and the subsequent fractions collected were assayed for neuraminidase and

HA activity (Fig. 18). Separation was not complete since no fractions

contained haemagglutinin activity with no contaminating neuraminidase.

However some fractions showed neuraminidase activity but no detectable HA.

Fractions 27-31 were pooled as a 'HA pool' and fractions 35-37 as a

'neuraminidase pool'.

The two pools were precipitated with 57. TCA and after treatment with

SDS and mercaptoethanol, the two pools, the pellet and supernatant from

the Tween 20 treatment and original virus were each analysed by poly-

acrylamide gel electrophoresis. Table 11 shows the area under the curve

t30 — 15

— Ei w

too at

I - 5

5 V IS

fraCtX,C11nIll

101

FIGURE: 15

Separation of RN!) from other virus matoria/ by oucr000--

dooxycholato 7,ralibnt contriFugatiun. Mathod is daseritsd

in toxt. Vireo: Herta. Migration from right to left.

Histogram in tha reciprocal of the HA titre.

102

FIGURE 17

03

..\.,..,..„ ..... ...

OD 0.2.1

0. .

0.0 . ■ 240 -260 280 300

nm. wavelength

Spectrum of isolatcd riMP.

103 TABLE 10

Identification of nucleocapsid polypeptide

Band 7 Migration of bromophenol blue marker

Gel 1 (virus)

Gel 2 (RNP)

10.0 mls

I 45.2

Mb

II 60.6

59.5

III 70.0 ••

—50

4 4 Z5.

10

—40

!

—30 ri;

4

20

,-10

WAD VOLUME

60

50 ►

40 E

a 30

0. 2

20

10

104

F/GURE 18

70

10 20 30 40 50

Fraction Number

Separation of virus (Ulster) subunits on a G200 sophadex

column after disruption with Teflon 20 at pH 10. The methods

used are described in the text.

0 Neuraminidase activity (pg NANA released).

■ Reciprocal of the HA titre.

Protein, pg.

105

of the peaks after a densitometer trace of the stained gels. Figure 19

shows the densitometer trace of a photograph of the five gels. The gel

of the neuraminidase pool, which had no detectable HA activity, had

only one band discernable by eye although the densitometer detected

another minor peak. The major peak of the neuraminidase pool had the

same migration as peak III in the whole virus gel, while the minor peak

migrated to the same position as band I of the whole virus. The 'HA

pool' which also had neuraminidase activity showed two peaks, corresponding

to I and III of the whole virus, the larger peak corresponding to I.

These results suggest that band III of the whole virus is the

polypeptide associated with neuraminidase activity while band I is

associated with the HA activity.

Other methods used to separate Tween 20 - released PA and

neuraminidase, sucrose density gradient centrifugation and DEAF:

chromatography produced similar results, complete separation of Hal and

neuraminidase was never obtained.

It is interesting to note that polypeptide I had a slightly lower

migration rate when treated with SDS in the presence of Tween 20 while

on the removal of Tween 20 it reverts to its former rate (Fig. 19).

1c

TABLE 11

Separation of haemagglutinin and neuraminidase

Area under curve (mm2) of densitometer trace

I II III REST

Virus (200dug) 997 1917 1450 374

Pellet 861 3817 1541 1282

Supernatant 1334 1570 1383 665

HA pool (50)ug) 573 - 495 -

N pool (20/ug) 8 - 142

N pool* 177 - 1055 OD

* Deflection across trace 0.2 OD. All others 1.0 OD.

107

FIGURE 19

ik"•-•N___"_,, ki

vows

PELLET

%....__,_...

SUPERNATANT

NEURAM IN I DASE ...........",... POOL

kr

HA POOL

IL.1I 1` 1 2 3 4

5

Migration au.

Separation of virus fractions, obtained by Tween 20 treatment

and G200 Sephadex chromatography, by acrylamide gel alectrophoresie

after SDS-mercaptoethanol treatment.

0 0

108 C. NEURAMINIDASE STUDIES

1) Relationship of neuraminidase to HA and infectivity

In order to determine if any strains possessed more neuraminidase -

than others the neuraminidase activity was compared with HA titre

(Table 12). HA was taken to be a measure of viral surface area and

preferred to a particle count because of the pleomorphism of the virus.

The results show that the ratio of neuraminidase activity to HA units

is generally constant, except for strains Beaudette C and, to a lesser

extent, Ulster which have considerably lower ratios.

The relationship between HA, neuraminidase and infectivity was

also investigated. The HA/EID50 and the neuraminidase /EID50 ratios,

again with the exceptions of Beaudette C and Ulster, even after allowing

for their high HA titres, were very similar for all strains (Table 13).

2) Kinetic studies of virus-bound neuraminidase

The amount of NANA released after 10 minutes was plotted against the

concentration of substrate used. All eight strains examined gave kinetics

curves essentially identical to that of strain Herts (Fig. 10).

The Michaelis-Menten constants (Km) were obtained by Lineweaver-Burk

plots (Fig. 20). All the strains had a Km of approximately 1 x 1e3M

(Table 14).

With fetdin as substrate the optimum pH of the neuraminidase of the

different strains, though varying slightly from experiment to experiment,

always fell in the range pH5.1-5.3 (Table 15) and was considered to be

the same for all the strains tested. Typical curves of pH effect with

fetitn as a substrate are shown in Figs. 21A and 21B. A second minor peak

109 TABLE 12

Relationship between haemagglutinin and neuraminidase activity

Strain Neuraminidase activity/ml

HA/ml Neuraminidase/ HA x 10 3

Field Pheasant 38.0 1500 25.4

Herts 360.0 10240 35.0

Texas 87.5 2560 34.1

WaiWick 84.5 2560 33.0

Eastwood Notts 171.0 5120 33.3

Beaudette C 271.0 20480 13.3

Al 176.0 5120 34.4

F 74.5 2560 28.1

(ueensland 172.5 5120 33.6

Ulster 845.0 40960 20.6

110

TABLE 13

Relationship between infectivity, haemagglutinin and neuraminidase activity

Strain HA

EID50 x 10-7

Neuraminidase activity (pH 6.0)

EID 50 x 109

Herts 2.9 8:5

Texas 3.9 7.3

Eastwood Notts 3.6 7.5

Beaudette C 19.6 22.2

B1 2.4 5.2

7 2.3 10.3

Ulster 10.1 14.2

60

20

0

111

FIGURE 20

Neuramine lactose (pmoles) 0 I 01 03

OA

1/s

Kinetics of the virus hound neuraminidaso of strain Herts.

Standard enzyme essay procedure was used except that the

concentration of neuramine lactose ues as above, In the

Linewoaver•Burk plot the units of Ve and 1/v Ewer moles/ml•

1:1.2 TABLE 14

Michaelis-Menten constant of neuraminidase at pH 5.4

Strain Km

Field Pheasant 1.0 x 10-3 M

Herts 1.0 x 10-3 M

Italien 0.9 x 10-3 M

Texas 1.0 x 10-3 M

Beaudette C 1.0 x 10-3 M

F 0.9 x 10-3 M

nueensland 0.9 x 10-3 M

Ulster 1.0 x 10-3 M

113

at pH6.3 was seen with all the strains tested except strain F. If

neuramine lactose was used as the substrate the pH optimum for all the

strains was raised slightly to pH5.4 (Fig. 21B), the second minor peak

was far more obvious although it still occurred at pH6.3. In these

experiments sodium acetate buffer was usually used for pH values from

4.1-5.5, phosphate buffer for pH5.6-7.2. If sodium acid maleate

buffer was used from pH5.1 to pH7.2 with both fetbin and neuramine lactose

the two peaks were present but with neuramine lactose the "minor" peak

was so much greater that it became the larger of the two (Fig. 21C and

21D).

In Table 15 the neuraminidase activity at physiological pH-:(07.2)

is shown as a percentage of the activity at the optimum pH. The strains

tested were generally in the range of 50-707. and although differences in

the strains were consistently found there was no relationship with

virulence.

3) Heat stability of virus-bound neuraminidase

The heat stability of virus-bound neuraminidase was estimated by

incubating virus suspensions at 56°C and measuring the remaining

neuraminidase activity at intervals. Within 20 minutes the neuraminidase

activity had been destroyed in all the strains tested except Beaudette C,

which still had 307 of the original activity (Fig. 22).

4) Effect of Tween 20 at pHIO

Treatment of virus with 17. v/v Tween 20 at pH10 caused the release

of neuraminidase with all the strains tested; between 70-100% of the

remaining activity was present as free neuraminidase that could not be

sedimented (Table 16). The total yield after treatment varied considerably

from strain to strain. With strains Herts and Beaudette C there was an

807. loss of neuraminidase activity while with Field Pheasant there was an

increase of nearly 407..

114

TABLE 15

The effect of pH on neuraminidase activity using fetuin as a substrate

Strain pH optimum pH of minor % of optimum peak of activity neuraminidase

activity at pH 7.2

Herts 5.1 - 5.2 6.3 53.4

Texas 5.1 - 5.2 6.3 60.0

Beaudette C 5.2 - 5.3 6.1 69.0

F 5.2 - 5.3 No minor peak 62.5

Ulster 5.1 - 5.2 6.3 49.7

I

a

Neu

ram

insd

ase

12

12

0

115

FIGURE 21

5

7 5

7

p H

The affect of pH on virus-bound neureminidaso undor different

conditions,

The normal assay procedure was used with the following

variations: Asistrain: Harts; buffers pH 4'2 - 5'5, O'lrl

sodium acetate-acetic acid buffer, pH 5'6 • 7'21 OiM phosphate

buffer; substrata: fetuin. 8.strain: Harts; buffer: acetate/

phosphate; substrate: neuramine lactose. C.strain: Ulster;

buffer: acetate/phosphate ( ); 042M sodium acid malaite

buffer (o )1 substrates fetuin. 0-strain: Herta; buffer:

sodium acid meloate; substrates neuramine lactose.

Neu

ram

inid

ase

0

to

1 00

80

50

40

20

116

FIGURE 22

0

5

10

15

20

Exposure t i me ( min )

rioA stability of virus-bound n,:uraminidnso ;-2t 560 C. t

tha spocifi,:d Urns 0•1m1 of purifi2d virus suspension was

added to N3m1 of ice-cold buffer, to stop further heat in-

activation, and then assayed as described in the text.

• Harts

• Texas

• Oeaudette C

O Queeneland

O Ulster

117 TABLE 16

Effect of Tween 20 (1% v/v pH 10) on virus-bound neuraminidase

Strain

Field Pheasant

7. activity of total control activity

control treated

Supernatant 10.0 125.0

pellet 90.0 13.3

Herts

supernatant 3.0 16.7

pellet 97.0 0.0

Texas


pellet 95.9 8.5

Beaudette C


pellet 90.0 2.2

Queensland


pellet 91.7 12.1

Ulster


pellet 98.3 7.6

'Control' samples were incubated overnight at 37°C with bicarbonate

buffer pH 10. For 'treated' samples the buffer contained 1% v/v Tween 20.

All samples were spun at 45,000 xg for 60 min, the pellets resuspended to

volume and the solutions tested for neuraminidase activity.

118

5) Neuraminidase activity in infected chorioallantoic membranes

The levels of neuraminidase per/mg. protein in the chorioallantoic

membranes:- of eggs infected with different strains appear to be related

to the virulence of the infecting strain, (Table 17). The plaque-

forming strains, i.e. Eastwood Notts and the more virulent strains,

show a linear relationship between cell associated neuraminidase and

MDT. The less virulent strains show less marked differences in

neuraminidase levels, presumably because there is very little

accumulation of the enzyme and much of the activity recorded is due

to whole virus particles associated with the membrane.

119

TABLE 17

Cell associated neuraminidase levels in infected chorioallantoic membranes

22 hrs after infection.

Strain Neuraminidase activity/mg protein

MDT (hours)

Herts 11.1 49

Texas 7.3 55

Warwick 5.9 62

Beaudette C 4.6 62

Eastwood Notts 2.8 70

B1 1.5 117

F i_16 119

Queensland 0.83 00

Ulster 0.5 co

120 D. CELL FUSION (FROM WITHIN) AND HAEMADSORPTION

Haemadsorotion

The adsorption of chick red blood cells to chick embryo fibroblast

cells infected with NDV was first seen 4-6 hours after infection. Figure

23 shows typical early haemadsorption to cells infected with strain

Texas. The *virulent strains, in contrast, show very little haemadsorption

until 8 hours or more after infection. RBC that become attached to

cells infected with virulent virus a few hours after infection always

attach at a central area of the cells (Fig. 23), whereas with avirulent

strains haemadsorption *Bays appears' at the cell extremities (Fig. 24).

The frequency with which this difference occurs suggests that this is

not merely the same effect viewed from a different angle. The virulent

strains examineds Herts, Warwick, Texas and Beaudette C; show

haemadsorption which increases exponentially until about 12 hours after

infection, by which time cells were almost totally covered by RBC and

syncytia had appeared. Massive cell fusion and haemadsorption caused

by Herts 16 hours after infection can be seen in Figure 25.

A comparison of haemadsorption by cells infected with Texas and

Ulster 22 hours after infection is shown in Figures 26 and 27. Infection

by strains F, Queensland and Ulster always produced far less

haemadsorption than cells infected with virulent strains, rarely

exceeding an average of 10 RBC/cell even after 36 hours.

The number of RBC attached per cell at different time after

infection was also estimated (Fig. 28). Haemadsorption occurcat a

much greater rate and is more extensive after infection with virulent

strains than with avirulent while Beaudette C which is often classified

as mesogenic appears to be intermediate to the two levels.

121

FIGURE 23

Haemadeorption by chick embryo coils 4hr. after infection

with strain Texas. Stains May GrUnwald•Giemsa, Magnifications

X1800.

FIGURE 24

•

a

Hasmnisorption by chiek embryo calls 10hr. after infection

with strain F. Stain: May GrUnwald-Giemsa. Magnification X1300.

r J

123

FIGURE 25

Haemedaorption by chick embryo cells 16hr. after infection

with strain Herts. Steins Noy Granwald.Giemse.Magnification X750.

FIGURE 26

Heemedsorption by chick embryo cells 22hr, after infection

with strain Texas. Stein: acridine orange. MegnificationiX440.

1 Jr

FI2URE 27

Hsanadsorption by chick embryo cells 22hr. after infection

with strain Ulster. Stainsacridine orange, Magnification X530.

cv 9 a 8

7 6 5

30

20 O

10

0

a)

E

ery

th

rocy

tes

• .0"

• •

•

5

-0-

••••• .60.•

"act—

1 6

FIGURE 28

4 8 12 16 20 24 28 32 36

Hr after inoculation

Haemadsorption by chick embryo cells infected with different

strains of NOV. At least 50 infect ,:d culls were observed for

each point.

• Harts

• Texas

• Warwick

• Beaudette C

0 r

• Queensland

A ulster

19 7 Difficulty was experienced in estimating attached RBC at 12 hours

or more after infection with virulent strains, because of the large numbers

of RBC sndi-the cell fusion that had occurred. However by seeding cultures

with low concentrations of chick embryo cells, infecting before

confluent monolayers were formed and by counting each nucleus in a

syncytium as one cell this problem was to some extent overcome.

No haemadsorption was seen in uninfected cells (Fig. 29).

2) Cell fusion (from within)

The ability of NDV strains to form syncytia in infected cultures

coincided directly with their ability to form plaques (Table 18). The

avirulent strains tested never formed plaques or syncytia, with the

exception of strain F which occasionally formed small syncytia. Cell

fusion was generally in evidence by 8 hours after infection although

with strains Herts and Texas small syncytia could be seen somewhat

earlier. Large syncytia containing up to 20 nuclei could be seen in

Hertz infected cell cultures by 14 hours (Fig. 30) although with other

virulent strains fusion to this extent took longer. Figure 31 shows a

large syncytium formed by Warwick 22 hours after infection. By 24 hours

after infection with Harts or Texas massive cell fusion had occurred

and the monolayer was almost completely fused into a single syncytium

(Fig. 32).

The size of syncytia formed appeared to vary from strain to strain

and, by counting the number of nuclei in each polykaryocyte at 12 hours

after infection the size was shown to be directly related to the virulence

of the infecting strain (Table 18). Occasionally a few binucleate cells

were present in uninfected cell monolayers, but no syncytia were ever

seen (Fig. 29).

1w8

FIGURE 29

Uninfected chick embryo calls. :twin: ley :rdnweld-Giemsa.

.Magoinciltions X430.

129

TABLE 18

Comparison of the ability to form plaques and syncytia in chick embryo

cell cultures infected with different strains of NDV.

Strain Presence of:

Plaques

Syncytia Average number of nuclei per syncytium

Herts ++ 16

Texas ++ + 15

Warwick -H- + 10

Beaudette C + + 8

4

rueensland

Ulster

The numbers of nuclei per syncytium were estimated from counts on at

least 50 syncytia 12 bra after infection.

190

FIGUiiL 30

Cell fusion in chick wiouryo calls 14hr. afGar infection

wiLh strcin Hares. Stains flay Grunwald- Glsmsa. Magnifications

X 700.

Syncytium soon in chick etriryo call culture 22hr. after

infection with strain Uaruick. Stain: r:y Grunwald-Ciemsa.

Magnification: X 700

*12111 ", *41".

•

(11

9

•

132

FIGURE 32

Massive cell fusion of chick embryo cells 24hr. after If

infection with strain Herts. Stain: May Grunwald..Giemsa*

Magnification: X 260.

133 3) Effect of inhibition of protein synthesis on virus induced

cell fusion and haemadsorption

The effect on haemadsorption and cell fusion (measured at 8 and

15 hours post infection respectively) of different concentrations of

2.-fluorophenylalanine (FPA) after infection by strain Herts is shown in

Figure 33. Fluorophenylalanine inhibits protein synthesis by replacing

phenylalanine to form non-functional proteins, 400,ug/m1 FPA inhibited

the level of both haemadsorption and cell fusion to as little as 10%

of that in untreated cells.

Cycloheximide also inhibits protein synthesis, with this drug

however the inhibition is due to interference to the messenger RNA

• ribosome complex (Mahler and Cordes, 1966). Cell protein synthesis

in uninfected cells, haemadsorption and cell fusion are all inhibited

to the same extent by cycloheximide (Fig. 34).

The inference from these experiments is that after infection

protein synthesis of a functional protein, or proteins, must occur

if NDV-induced cell fusion or haemadsorption are to subsequently

develop.

4) The effect of time of addition of FPA on cell fusion and haemadsorption

Fluorophenylalanine (500 pg/ml) was added to chick embryo cell

cultures at different times after infection with strain Herts, and the

effect on cell fusion and haemadsorption (measured at 15 and 8 hours after

infection respectively) was estimated (Fig. 35).

Both cell fusion and haemadsorption were almott completely inhibited

if the FPA was added within 2 hours of infection, but were unaffected if

added later than 4 hours after infection. This suggests that the protein

or proteins that are necessary for these phenomena must be synthesised

within 3 hours of infection.

• •

• A

• • •

134

FIGURE 33

100

0

0

80

a 60 4.) CU nd

_C -C 4-0 CL

0 0

0 40 4-

0 ld

0 20

<r) 0

100 200 300 400

Fluorophenylalanine (fig./m1.)

The effect of different concentrations of fluorophenylalanine

on haemadsorption (A ) and cell fusion (m), by chick splay*

cells infected with strain Herts. Fluorophenylalanine was

added ihr. after infection.

135

FIGURE 34

100

15

lEj • 80

C W

rt E _c • R 60 (1.)

O -c O

*,;°

40 5 0

> _c • . _

4 14C

0

20

5

10 15

20

Cycloheximide /mI.)

Effect of difforsnt concontrationl of cyclohoxinido on protein

synthesis in uninfoctad chick embryo coils ( • ) and on hr!en-

adsorption ( A) and cell fusion (is ) in chick embryo cells

infected with strain Herts. Cycloheximide was added lhr.

altar infection. Protein synthesis W33 measured in uninfected

cells 3hr. after the addition of cycloheximide by the uptake

of 3144suoino into en acid-insoluble fraction.

136

FIGURE 35 -

0

C 0

C

0

r3

0 • 80 C

ed

• 60 _c 0

0 40

0

20

0

0 4 8 12

16 Hr after infection

Effect of ceding fluerophanylelanine (5031pgim1) at different

iron after infection on heemadeorption (A) and cell fusion

(a) in chick embryo cells infoctod with strain Herts. The

points indicate the times after infection at which fluorc»

phenylelenine was added.

137 E. INHIBITION OF CELLULAR PROTEIN SYNTHESIS

1) Effect of infection with different strains on cellular protein synthesis

The effect of different strains of NDV on cellular protein synthesis

was measured by the ability of chick embryo cells to incorporate' 3H-leucine

in the trichloracetic acid - insoluble material after a 30 minute pulse.

In Figure 36 incorporation of radioactive label is expressed as per cent

of incorporation into uninfected cells. The virulent strains Herts,

Warwick, H, Texas, Field Pheasant, and Beaudette C all caused marked

inhibition of protein synthesis, 50% at 6-8 hours after infection and

as much as 90% by 12 hours. The vaccine' strain F inhibited prOfein

synthesis by 40% but this level was not reached until 18 hours after

infection. The avirulent strains Queensland and Ulster had very little

effect on protein synthesis.

The virulent strains also cause a marked cytopathic effect in chick cells

and there is a possibility that inhibition of protein synthesis is due

solely to the death of cells. Disruption of the cell membranes and

lysis of the cell is associated with cell death. The degree of cell

death can,. therefore, be measured by the release of a substance, normally

found solely in the cells, into the supernatant. The enzyme lactate

dehydrogenase (LDH) is such a substance and the measurement of release

of LDH activity after infection with Herts, Texas, Queensland and F was

measured and expressed as a percentage of total cellular lactate

dehydrogenase activity. A slow loss of LDH activity was recorded

during the first 16 hours after infection with all the strains. After

this time a rapid increase was seen only in cells infected with virulent

strains. These results suggest that gross cellular damage induced by

virus infection occurs after 16 hours and could not, therefore, account

for cell protein synthesis inhibition which occurs much earlier.

O 0 00 • • 100

80

3 • 60

1 40

20

0

19 8

1

I 9 6

V

40

:4 • 20

g 0 120

0

100 U

• 80

o ▪ 60V

40

20 0

,

FIGURE 36

1 I I -

I-

0- 0

I I i HERTS

•-•

o

I

o

I

o-'

1 I 1 1 1 TEXAS

o

0

I I I I I H -

_

...:

...:

-

- WARWICK.• BEAUDETTE C FIELD PHEASANT

-

-

_

00-0/

1 1

QUEENSLAND

0

0/

/ 0

1 1 1 1 1 1

ULSTER

1 1 1 1

ea............ F\,...:

-,

0-0

,0''

0.0/ I I I I 4 8 12 16 20 24 4 8 12 16 20 24

Hr after infection 4 8 12 16 20 24

Protoin Synthosis inhibition by different strains of NOV.

Protein synthesis in infected ombryo cells wen measured by

tho uptake of 3H-leucine into a TCA insoluble fraction and

axpressed us incorporation into uninfected calls (40.

The releaso of LDH ftorn chick embryo cells infect©d with WV

strains (o) is oxprossed as 5 total collulnr LDH.

139 2) Effect of p-fluorophenylalanine on virus induced inhibition of

cell protein synthesis

Two strains were examined - Harts and H - to study the effect of

FPA on virus induced cell protein inhibition. One hour after infection

FPA at various concentrations was added to cell cultures. Seven hours

later cultures were pulsed for 1 hour with 3H-leucine and incorporation

into a TCA-insoluble fraction was measured. At this time control cells

with no FPA show 60% inhibition of cell protein synthesis. The results

obtained (Fig. 37) show that FPA decreases the amount of cell protein

synthesis inhibition, in the presence of 400 ug/m1 FPA cell protein

synthesis is inhibited by only 107. These results indicate that like

cell fusion and haemadsorption protein synthesis is necessary for the

virus induced inhibition of cell protein synthesis.

3) Effect of time of addition of FPA on virus induced inhibition

of cell protein synthesis

Cells were infected with strain Herts and at intervals after infection

500 ug/ml FPA was added. At 10 hours after infection the cultures

were pulsed for 1 hour with 3H-leucine and the extent to which cellular

protein synthesis had been inhibited was estimated (Fig. 38).

The addition of FPA within 3 hours of infection completely reversed

the inhibition of cell protein synthesis but the later the FPA was

added after 3 hours the nearer the level of inhibition approached 707.

which was the amount of inhibition obtained with infected cella in the

absence of FPA. These results suggest that a protein, or proteins, ,

synthesised within 3 hours of infection is responsible for. the

inhibition of cell protein synthesis.

-

FIGURE 37

140

100

ii7; 80

60 •_

40 0

•_ 20

:E

100 200 300 400 Fluorophenylalanine (pg./ml.)

Prevention by fluorophonylplanine of tho inhibition of

cellular protein synthesis in chick embryo cells by strains

Horto (•) and H (0). Fluorophsnylnlenine was added ihr.

after infection to control and infected cultures: 7hr. later

cultures were pulsed for lhr. with 3H-loucine and incorporation

into an acid-insolublo fraction wee measured.

141

FIGURE 38

100 0

•V go .c

C 60

15 0 CL

40 C 0

20 C

1 2 3 4 5 6 7 8 9 10 Hr after infection

Effect of adding fluorophonylalanina (50Gpg/m1) at different

time; after infection on the inhibition of collular protein

synthosis by strain Herta. At intervals after infection fluoro-

phanylalanine was added to infected cultures and uninfected

control cultures. All cultures ware pulsed with 31-6.1eucina

for ihr"agicTr. infection and incorporation into an acid-insoluble

fraction measured.

142 F. EFFECT OF CONGO RED ON THE MORTALITY OF EGGS INFECTED WITH NDV

The effect of congo red on the virulence in eggs of 5 strains:

Herta, Texas, B1, Queensland and F, was tested. Each of the virus

strains was inoculated into the allantois of 10-day-old eggs as a

high dose, . 5 x 108 EID50, or as a low dose, 1-50 EID50. Congo red

was inoculated immediately prior to infection. The mortality of

uninfected eggs treated with conga red was not greater than 57. at

168 hours after inoculation.

Up to 72 hours after infection treatment with congo red gave

considerable protection to eggs infected with low doses of the two

virulent strains (Fig. 39). In later hours, however, the mortality

reaches a similar level in both treated and untreated eggs. Initial

protection suggests that prevention of release does not enable

infection of sufficient cells to cause the death of the chick embryo.

High mortality at all, although greatly delayed, is presumably due

to infective particles being released by death and Lysis of originally

infected cells and eventual infection of sufficient cells to cause

death of the embryo.

Congo red has very little effect on the death of eggs infected

with high doses of virulent virus (Fig. 40). This is predictable

as high doses of virus would ensure sufficient\cells are infected and

killed to bring about death of the embryo even without release of

virus particles.

Eggs infected with low doses of strain B1 and Ulster in the

presence of congo red showed very little variation from infected eggs

without congo red (Fig. 41). Eggs infected with Queensland in the

presence of congo red however produced up to 407. mortality within 96

hours. There is no apparent explanation for this although repeated

experiments produced similar results.

143

High doses of avirulent (or low virulence) virus produced high

mortality of eggs in the presence of congo reds 80-907. in 96 hours

and 1007. in 168 hours compared to 20-307. at 96 hours without congo

red (Fig. 42). This suggests that the prevention of release of

virus can bring about the death of an embryo if sufficient numbers

of cells are infected, even if the virus strain is normally

avirulent.

It should be noted that embryo mortality in the presence of

congo red occurs at a much higher rate after virulent infections

than with avirulent infections (Figs. 40 and 42).

60

cs-P N 0 0 u, 20

0 >- 1- = 4 o-. cc

2 0100

60--

20 1 96

1 HE RTS

I fl NI' 24 48 54 72

HR. AFTER INFECTION

100

i 1 FIELD PHEASANT

144

FIGURE 39

Virulent_virus: low inoculum

Virus w-s inoculated at the following levels: Herta 50 EIDro

per egg; Field Pheasant 12E/050 per egg. Congo red (1mg in

0•25m1) uns added immedistely prior to infection (closed

histogram) control eggs received 0•25m1 sterile distilled

water prior to infedtion(open histogram). NR: not racorded.

X.5

FIGURZ 40

100

60

o 20 w 0

LL

' *Tr I- 100 O cr

2

60

20

HERTS.

FIELD PHEASANT

24 40 48 54 72

96

HR. AFTER INFECTION

Virulent virus: hi:-41 inoculum

Virulent virus was inoculated at the following levels:

Harts 2+5 x 109 EID50 per egg; Field Pheasant Gx 100 CID50 per egg. Congo red (1mg in 0•25m1) W13 added immediately

prior to infection (closed histogrcm), Control eggs received

0,25m1 of distilled water prior to infection (open histogram).

Mn: not recorded,

146

FIGURE 41

0-12

100

60

20

81

I I I H.

U. 0

M

OR

TA

LIT

Y

100

60

QUEENSLAND

20

24 48 72

96

HR AFTER INFECTION

Low virulence: low inoculum

Virus of low virulence was inoculated at the following levels:

B1 20 EIDSO per egg; Queensland 3EI050 per egg; Ulster

15 EID 50 per egg. Congo red (1mg in 0:25m1) was added immed..

iately prior to infdction (closed histogram). Control eggs

received 0.25m1 of sterile distilled water prior to infection

(open histogram). No deaths occurred amongst eggs infected

with strain Ulster.

B1

mo—

60 -

1

-

20

QUEENSLAND

c.t) LI — I,

R. NR rl fl N R

ULSTER

MO

RTA

LITY OF 60-

20 -

100 -

60 -

20 - NR

147

FIGURE 42

24 48 54 72 96

168

HR. AF TER INFECTION

'ow viyulonces high inoculum

N R

Virus of low virulence was inoculated at the following

levels; 81 1x109 EIDSO per egg; Queensland 1•6x1010 EID50

per egg; Ulster 3x1010 EIDSO per egg. Congo red (1mg in

0.25m1) was added immediately prior to infection (closed

histogram) control eggs received 0625m1 of sterile distilled

water prior to infection (open histogram). NRs not recorded.

148

G. THE SYNTHESIS OF NDV-INDUCED PROTEINS

The synthesis of virus-induced proteins in NDV-infected cells

could be detected iising the double labelling technique described

in the methods section. The extracted proteins gave the pattern

seen in Figure 43 after polyacrylamide gel electrophoresis and

staining with amido black. Since total cell protein has been

separated on the gel there are very many bands, so many in fact

that it would be impossible to distinguish the extra bands induced

by infection with virus and demonstrates the necessity for labelling

the virus-induced proteins.

All proteins in uninfected cells labelled with 3H-leucina have

complementary proteins in 14C-leucine labelled uninfected cells.

The graph in figure 43 shows the ratio of 3H to "C DPH in a sliced

gel after electrophoresis of a double-labelled uninfected cell

preparation, as expected there are no major peaks and the proteins

show an almost constant ratio of 3 H/14 C. In infected cells, where

virus-induced proteins labelled with 3H have no complementary 14C

labelled proteins in uninfected cells, obvious peaks of 3H/14C ratios

can be seen (Figs. 44-48).

By comparison with egg-grown purified virus run in parallel with

the double-labelled cell extracts it can be seen that the three major

structural proteins of NDV could be detected from the second hour

(1-2 HR) in cells infected with Herta and Field Pheasant but not

until the 4th hour (3-4 HR) with strain Queensland. In the second

hour structural proteins I and III were detectable in Beaudette C

infected cells but protein II was not, although it appeared in the

4th hour.

149

FIGURE 43

3H/ 14c 2

UNINFECTED CELLS

•••••••1

1

••••••V••••••W1/4.••••••••••••%...vii....

10 20 30 40

50

Migration(mm.)

Polyacrylamide gel electrophoresis of polypeptides extracted

from 3H-lewcine and 14C-leucine uninfected cells. Cells wore

pulse-labelled for one hour.

The photograph shows a typical pattern obtained after

electrophoresis and staining pulypeptides extracted from chick

embryo cells whether infected or not.

1 1 1 1 A

—

.—

I I

/ I I[

?I

i

I

1 1 HERTS III

—

—

—.

I I

—

I I I

FIELD PHEASANT

—

I I BEAUDETTE C

1--

.— .... I 1 I I 1

QUEENSLAND

...- —.

1.4.011VPHII4844••••NMIORResemeeseVeliP0 ....., I I I 1

A I Ix

— • • •

—

—

I%

III mum) • —

—

—

gym I

2 3H/14c

2

B CPM 6

x10-2 4

0

3

ail .14 C 2

150

FIGURE 44

10 20 30 40 50 Migration(mm)

Induced proteins I-2hr. aftor infection.

Polyacrillamide gel electrophorosie of polypeptides extracted

from 3H-leucine labelled infected cells end 14C-leucine

labelled uninfected cells. Celle were pulee4abelled one

hour after infection for one hour. Purified 3H-loucine lab.

oiled virus (strain Queensland) was similarly extracted and

run in parallel for comparison (o ).

4c

3

2

BEAUDETTE C

3IY 2 1— 14c

151

FIGURE 45

QUEENSLAND

2 —311/ 'MC

1 —

1 10 2 3

114 5

Migration (mm.)

Induced proteins 3.'4hr. after infection.

Pelyacrylamide gel electrophoresis of polypaptides from

3H-leucine labelled infected cells and 14C-laucine labelled

uninfected cells. Cells ware pulse-labelled 3hr. after

infection for one hour. Similarly extracted purified virus

(strain Harts) was run in parallel, the migration of the

structural polypeptides detected by amido black staining are

indicated.

3

I 1 1 i 10 20 30 40 50

7

5

3

1

I

QUEENSLAND

152

FIGURE 46

HERTS

5

3

i

t

1

1 BEAUDETTE C

Migration (mm.)

Induced proteins 5.6hr. after infection.

Polyacrylamide gel electrophoresis of polypeptides extracted

from 3H.leucine labelled infected cells and 14C.leucine labelled

uninfected cells. Celle were pulse labelled 5 hours after

infection for one hour. Closed arrows show the migration

of structural polypeptides run in parallel (strain Herta).

I I I i 1 10 20 30 40 50

Migration(mm.)

153

FIGURE 47

Induced proteins 8-10hr. after infection.

Polyecrylamide gel electrophoresis of polypeptides extracted

from 3H.mleucine labelled infected calls and 14C-leucine

labelled uninfected cells, Celia were pulse labelled 8hr.

after infection (strain BeaudetteC) or 9hr. after infection

(strains Herta and Queensland) for one hour. Closed arrows

show the migration of structural polypeptides run in parallel

(strain Hefts).

,_.

1

•

ii, NSI

/1,

I

H2 '

/ 11

I M)111011ra%!

'I

I

1 I HERTS

-

f

NS2 -

1

I-

NS, 1 'I

/ i 1

BEAuDETTE C

NS2

-.

I I

NS,

- 1 1. 1

4.1j\rAWL1J1‘.14(

OUEENSLAND

-.

NS2

--

1.... I A I II ICE

• • VIRUS IBC)

31-1/ 14c t

314114c I

3H14c

10

5

4

2

0

CPM

ato-2

154

FIGURE 48

10 20 30 40

50

Migration (mm.)

Induced proteins 15.46hrs after infection.

Polyacrylamide gel electrophoresis of polypeptidss extracted

from 3H4eucine labelled infected cella and 14Cibleucine

labelled uninfected cells. Cells wore pulse-labelled 15hrs

after infection for one hour. Purified 3H-leucine labelled

virus (strain 3eaudetto C) was similarly extracted and run

in parallel ( o ).

155 The structural protein 1 which had a corresponding protein in

Queensland infected cells seems to be represented in Beaudette C and

Herta infections by protein '?I', although this protein_migrated further

than protein I of egg grown pure virus preparations of either of the

strains run on parallel gels (Figs. 44-48). This assumption is

further complicated however by the occasional appearance in some gels

of these strains of a peak showing the same migration as polypeptide

(Fig. 44 1-2 HR Beaudette C, Fig. 48).

Structural protein A which is not always discernable in gels

of pure radioactive labelled virus can be seen in all three of the

strains in the later samples.

Two non-structural proteins are produced which are common to

all three strains. The most prominent of these, NS, which has a

molecular weight of approximately 180,000 is first seen in all the

strains in the 6th hour (5-6HR'Fig. 46). The production of NS, in

Queensland infected cells is at a maximum at the 11th hour when it is

the most dominant peak (Fig. 47). The other non-structural protein,

NS2, has a molecular weight of approximately 36-38,000. The peak

produced by N52 was always very small, however, it is unlikely to be

adventitious as it was present consistently from the 4th hour.

Herts infected cells produced two non-structural proteins which

were not produced in cells infected with the other two strains.

Protein H1 was first'seen 9-10 HR.after infection (Fig. 47) and has

an approximate molecular weight of 200,000. in addition another protein,

H2' was detected in the 16th hour which has an approximate molecular

weight of 140,000 (Fig. 48 Herta 15-16). Smaller peaks at the same

migration as H2 were seen earlier in infection but were inconsistent

in both appearance and migration.

A summary of the results and the proteins induced in infected

cells is given in Table 19.

Strain and times seen

Protein

All strains and pure virus

All Queensland gels except 1-2 HR and in pure virus preparations virus

In all Herts and Beaudette C gels but not in pure virus preparations

In all'gels except 1-2 HR

III

A

I

NS1 In all gels after 3-4 HR. Not in pure virus preparations

Non-structural 180,000

If NS2 36-38,000 In most gels but not in pure virus preparations

111 200,000 Detectable in Harts 9-10 HR and Hertsl5-16 HR only

II H2 140,000 In Herts 15-16 HR only

156

TABLE 19

Summary of the induced proteins seen during NDV infections

Type Approximate molecular weight

Structural ) 200,000

Structural 88,000

Structural?? 78,000

Structural 58,000

11 42,000

157

H. RNA PRODUCTION OF INFECTED CELLS IN THE PRESENCE OF ACTINOUYCIN D

The production of RNA in chick embryo coverslip cell-cultures

infected with different strains of NDV was measured by the incorporation

of 3H-uridine into an acid insoluble fraction in the presence of 5 ug/ml

actinomycin D. Under these conditions only the production of virus

specified RNA is measured. The results show a remarkable correlation

between the amount of RNA produced and the virulence of the five strains

tested (Fig. 49). Strain Herts shows 2357. more uptake of 3H-uridine

than Queensland after 12HR, while Texas and Beaudette C show respectively

1857. and 1357. more at that time.

RNA production also appears to begin somewhat earlier with the

virulent strains than the avirulent.

The drop in RNA production seen in the more virulent strains,

notably Herts, appears to be due to loss of cells from the monolayers

as a consequence of the cytopatbic effect of the virus.

NDV RPM production

Chick embryo cells HE RTS

TEXAS

ULSTER

QUEENSL AND

CONTROL 0

...

BEAUDE TTE C

158

FIGURE 49

300

2 a. U

200 a

E 0

100

2

4

a

10

12

Hr after infection

NDV RNA production in chick embryo cells.

The accumulative uptake of 3H-uridine in the presence of

actinomycin 0 (Speml) into an acid-insoluble fraction was

measured at hourly intervals after infection of chick embryo

cell coverslip cultures. Results are expressed as a % of

Queenalanctrifacted cells after 12hr. (1001As approx. 3,000cpm.).

19 IV DISCUSSION

The object of this work was to investigate the wide range of

virulence seen between strains of NDV. Most of the sections of the

work studied have been previously suggested as, or shown in preliminary

experiments to be, likely candidates for the causitive agent of the

differences in virulence.

A. STRUCTURAL PROTEINS

Since the demonstrations by Ornstein (1964) and Davis (1964)

of the advantages of polyacrylamide gel discelectrophoresis over

other techniques used for protein separation this method has been

increasingly used in investigations into the structure of virions and

production of virus proteins in infected cells. In particular,

acrylamide gel electrophoresis has been used in the presence of SDS

after disruption of virions or treatment of other proteins with this

detergent, since under these conditions migration of proteins is directly

related to their molecular weights (Shapiro, Vifluela d Maize', 1967).

Although acrylamide gel electrophoresis of disrupted virus after

treatment with SDS is a powerful tool in the investigation of virus-

structural-proteins there are certain drawbacks to the method that

should be considered. The most obvious criticism is that more than

one polypeptide may be represented in any one band. There may also

be one or more components of the virion which are of particular importance

but present in such low amounts as to be undetectable by this method.

The three major and one minor polypeptide bands consistently seen in

this study should, therefore, be regarded only as the minimum number

of polypeptides likely to be of viral origin.

The exact correspondence of the peaks of radioactivity in gels of

labelled virus to the stained bands demonstrates that the polypeptides

160

are specifically induced by the virus, incorporation into the virions

of proteins made before infection would mean that stained bands of these

proteins would not show a corresponding radioactive peak. However,

there is no information as to whether or not the proteins are coded

for directly by the virus genane or are produced by the cell as a

response to infection. The results show (with the considerations

mentioned ) that the NDV virion consists of three major polypeptides

I, II and III and one minor polypeptide, A.

The validity of polypeptide II as a single or multiple polypeptide

is of particular interest. In this work, that of Evans and Kingsbury

(1969), Haslam et al (1969), Bikel and Duesberg (1969), Mountcastle

et al (1970) and Mountcastle, Compans and Choppin (1971), it has been

shown that isolated RNP migrates as a single band to the position of

polypeptide II of whole virus and it has been assumed that the RNP

polypeptide is the only polypeptide in this position. However

Mountcastle et al (1970) have also shown that the polypeptide of RNP

isolated from a tryptic digest of cells infected with NDV migrates with

a molecular weight of 47,000 to the position of polypeptide IIa in this

study. A band at this position was also reported as an occasional band

by most of the other authors. Presumably the partial digestion of the

RNP by trypsin is sometimes mimicked by SDS disruption causing the

appearance of the occasional band IIa. Another problem is the apparent

uptake of labelled glucosamine into polypeptide II - the band identified

as the RNP polypeptide. Using fluorescent antibody techniques RNP is

first detected in the cytoplasm of infected cells close to the nucleus

(Rott and Schiffer 1964, Rott and Scholtissek, 1967) and would seem

unlikely to be a glycoprotein, as glycoproteins are usually associated

with cell surface membranes. Mountcastle et al (1971) also show activity

161 at the same migration as polypeptide II, identified as RNP, after

labelling with radioactive glucosamine, however) they report that there

is no uptake of labelled glucosamine detectable in isolated RNP and

suggest that the results obtained are due to another polypeptide showing

identical migration to polypeptide II. If this is the case it is

remarkable that in the attempts to separate and isolate the sub units of

NDV both in this study and that of Haslam et al (1969) no further evidence

of this protein has appeared.

To identify a viral subunit that unit should,ideallyt be isolated

and purified. The lack of success in the studies undertaken so far, i.e.

this study and that of Haslam et al (1969) and Evans and Kingsbury (1969)

in separating the neuraminidase and haemagglutinin sub units has prevented

the absolute confirmation of the suggestions that polypeptide I is associated

with haemagglutinin, and polypeptide III with neuraminidase. However,

although different methods of disruption and analysis were used, similar

conclusions as to the nature of the polypeptides were reached in each

study, although Evans and Kingsbury (1969) do•:not associate neuraminidase

with polypeptide III. The fact that polypeptide I is a glycoprotein

gives more significance to the possibility that this band is HA since it is

known that haemagglutinin originates at the cell surface membrane

(Mott and Scholtissek, 1967). Failure to bring about separation of the

viral sub units in a biologically active form may well be a warning against

the general assumption that the activities are associated with separate

proteins, it is arguable that both activities could be associated with

one protein or with different polypeptides of the same protein.

The absence of any significant difference in the number, molecular

weight or proportion of the polypeptides, seen in this study, from one

strain to another suggests that differences in virulence are due to

differences that occur in the production of the virus between the

moment of entry and the moment of release.

162

B. NEURAMINIDASE STUDIES

None of the properties of NDV neuraminidase examined show any great

variation from strain to strain. Unlike influenza virus (Rafelson et al,

1963) it appears that different NDV strains possess neuraminidases which

are essentially identical in their biochemical properties.

On assessment of physical characteristics Beaudette C appears to be

the most atypical strain. The heat resistance shown by Beaudette C

neuraminidase is probably due to the protection of the intact virion since

Drzeniek et al (1966) have shown that neuraminidase isolated from Beaudette

C virus is just as heat labile as neuraminidase from the other strains.

Beaudette C has twice as much haemagglutinin as other strains which could

account for the protection of the neuraminidase, although estimations of

the proportion of polypeptide I is no greater in Beaudette C than other

strains. Rott, Reda and Schafer (1962) have shown that a large fraction of

the virus in their Beaudette C suspensions was devoid of ribonucleoprotein.

From the HA/EID50 ratios which are, in fact, estimations of non-infectious

particles, this does not appear to be a general characteristic of all the

strains, although Ulster also seems to have a high HA/EID50 ratio.

It is difficult to assess the significance of the differences in the

effect of Tween 20 on the neuraminidase of the different strains.

Presumably the enzyme of those strains showing a low yield of released 6tRel

neuraminidase after Tween 20 treatment is/more intimately associated with

the lipid membrane of. the virus, or that their enzyme is different in

some way and rendered inactive in the presence of Tween 20.

The pH optimum of NDV neuraminidase is lower than that recorded by

Drzeniek et al (1966) for Italien and Beaudette C. However this is

probably due to variation in the substrate, it has been shown by

Rafelson et al (1963) that pH optima of neuraminidase differ with

substrate. The observation of two peaks of enzyme activity has been

previously recorded by Drzeniek, Bagel and Rott (1967) with the neuraminidase

of bovine parainfluenza 3 virus. Since in both the substrates used the

163

NANA is not specifically attached i the two peaks could be produced by the

availability of substrate varying with pH. However, Drzeniek (1967) using

2-3' and 2-6' linked sialolactose, has shown that NDV neuraminidase,

unlike Vibrio cholerae neuraminidase cleaves only 2-3' linkages and that

- the failure to cleave 2-6' linkages i* unaffected by pH changes.

Alternatively the enzyme may exist in more than one form, with different

pH optima.

The relationship between virulence and cell-associated neuraminidase

levels has been noted previously by Reeve et al (1970) who have shown

that all major virus antigens accumulate in the chorioallantoic membrane

of eggs infected with virulent strains but not in eggs infected with

avirulent strains of NDV. Accumulation of neuraminidase and other antigens

in virulent strains infers that there will be a far more extensive

modification of the cell membrane than with avirulent strains and this

alone could cause cell death. However, it is also possible that cell

death could be brought about by generalised disruption of cell membrane

caused by the accumulating neuraminidase, or by the action of neuraminidase

at a specific site or sites in the cell (Bang 1953, Allison and

Mallucci, 1965).

164

C. CELL FUSION AND HAEMADSORPTION

1. Haemadsorntion

Bang (1953) reported that avirulent stains were produced at the cell

surface on microvilli while virulent strains appeared to originate more

within the cytoplasm. This may well account for the observation in this

study that intial haemadsorption to cells infected with avirulent strains

is at the extremities of the cell but closer to the nucleus with virulent

strains.

Banhowski (1964) had previously shown that cells infected with

avirulent strains of NDV took longer to show haemadsorption than cells

infected with virulent virus, in the present study it is demonstrated that

haemadsorption is a direct consequence of the virulence of the infecting

strain, at least on the basis of gross virulence, i.e. velogenic

mesogenic and lentogenic strains. The extensive modification of cell

membrane, demonstrated by haemadsorption, could in itself be responsible

for the death of cells and ultimately the virulence of the infecting

strain. It is particularly noticeable that avirulent strains appear

never to reach the levels of haemadsorption attained by virulent strains,

increase in red cell attachment is negligible between 16-36 HR after

infection by avirulent strains. The differences in haemadsorption could

be due solely to a difference in rate of virus production but this does

not explain the evidence which suggests that virus is released at the

same rate to similar levels from all strains (Reeve and Waterson, 1970,

Liu and Bang, 1953, Pennington, 1967). It seems likely that differences

in haemadsorption are related to the accumulation of virus antigen recorded

by Reeve et al (1970). However this too may be the result of a different

rate of production.

2. Cell fusion

Cell fusion in chick embryo cells was also shown to be directly

related to the virulence of the infecting strain, the most virulent

165

Mit.• 0...40 J.

strains producing the largest polykaryocytes. Similarly only strains

which produce plaques showed cell fusion to any great extent, which

may suggest that cell fusion is an initial stage in plaque forma.

The relationship of cell fusion to virulence has been reported with

other viruses (Plowright, 1962, Bodon and Greczi, 1966, Ilahmias and

Dawdle, 1968) but the reason why virulent virus and not avirulent should

induce cell fusion and the mechanism by which they cause it, remains

obscure. At the moment there are two main theories for the mechanism

of cell fusion (for review see Postei 1970). The first (Ejercito,

Kieff d Roizman, 1968) postulates that maturation and accumulation of

virus-products at the cell surface causes gross changes in the cell

membrane which result in cell fusion. In the NDV system it is known

that accumulation of virus products is important with respect to virus

virulence and the vast haemadsorption to cells infected with virulent

strains is evidence of the massive cell modification that occurs.

However Poste (1970) disagrees with this theory on the basis that some

viruses that mature at the cell surface do not fuse cells. This is

exactly the case in the NDV system and yet only the strains that

accumulate, irus components and drastically modify the cell surface

fuse cells. The other theory, credited to Allison (1967), is

basically that modification of the cell surface glycoproteins which

must occur before cell fusion can take place is brought about by the

release of lysosomal enzymes at the cell surface (for review see Poste,

1970). This is supported by the findings of Allison and Mallucci

(1965), using NDV, they have reported that lysosomal damage and release

of enzymes occurs in virulent but-not avirulent infections. However

this explanation merely shifts the question one step since there is

no explanation of why virulent virus infections cause lysosomal

damage.

166

3. EFFECT OF INHIBITION OF PROTEIN SYNTHESIS ON CELL FUSION AND HAEMADSORPTION

The results obtained of the effect on haemadsorption and cell

fusion of FPA and cycloheximide suggest that not only is protein

synthesis necessary for the manifestation of these phenomena but that

inhibition after 4 hours post infection has little effect on their

production. This suggests that the proteins responsible for both cell

fusion and haemadsorption are produced in sufficient quantities in the

first 4 Hitt° render further synthesis of these,proteins unnecessary

for maximum occurrence of cell fusion and haemadsorption. Or, in the

case of haemadsorption, that sufficient protein (i.e. haemagglutinin)

is produced in the first 4 hours to produce maximum modification of

the cell membrane.

The inhibition of cell fusion by inhibition of protein synthesis

has previously been reported with other viruses. Kaku and Kamshbraa

(1964) reported the inhibition of cell fusion in vaccinia infected L-cells

in the presence of puromycin and Falk* (1965, 1967) showed that fusion

by harps* simplex virus of rabbit kidney cells was prevented by the presence

of cycloheximide and FPA but not'chloramphenicol.

The nature of the proteins or protein that are responsible for

cell fusion by NDV is not blear. The fact that high multiplicities of

virus can fuse cells without protein synthesis (cell fusion from without)

and the inhibition of both types of cell fusion with NDV specific

antiserum (Brett and Gallaher, 1969) suggests that a structural protein

may be implicated by its action at the cell surface. Although it is

difficult to visualise this in terms of the 'lysosome labilisation'

theory of cell fusion already discussed. In fact the failure of

cells to fuse in the presence of specific antisera is excellent

evidence that the maturation of the virus at the cell surface is

important for cell fusion.

167 D. INHIBITION OF CELLULAR PROTEIN SYNTHESIS

The results show that inhibition of cell protein synthesis by NDV

strains was directly related to the virulence of the infecting strain.

The results of LDH release from infected cells suggest that the inhibition

of cell protein synthesis is not merely a consequence of cytopathic

effect, this conclusion was also reached by Wheelock and Tamm (1961)

using Hela cells.

Scholtissek and Rott (1965) reported that after 10 hours chick

embryo fibroblast cells recovered from the initial inhibition of protein

synthesis induced by strain Italien. There is no evidence, in this

investigation of any such recovery after inhibition by any of the

strains used. Similarly Wilson (1968) using Texas and Beaudette C

failed to show recovery.

Inhibition of cell protein synthesis by other viruses has also

been recorded (Martin and Kerr, 1968). In particular the picornaviruses

and vaccinia virus show inhibition (Martin and Kerr, 1968, Bablaniin,

1970) which is particularly rapid (within 3 HR of infection) and seems

unlikely to be comparable to the slower inhibition by NDV virulent strains.

The experiments using FPA to inhibit functional protein synthesis

demonstrate that a protein (or proteins) synthesised in the first 3 HR

after infection is responsible for the inhibition of cell protein synthesis.

This infers that the genom6 of avirulent strains is unable to code for

a protein (or proteins) with this ability.

It is unlikely that the inhibition of cell protein synthesis, or of

all macromolecule synthesis, is alone responsible for the difference

in virulence and cytopathogenicity between strains of NDV. Neither

puromycin, which inhibits protein synthesis, or actinomycin, which

prevents RNA synthesis cause the death of Kreb's ascites or Hela cells

in less than 24 HR (Martin and Kerr, 1968); while control chick fibroblast

or BHK cells treated with actinomycin D azauridine or FPA showed no

sign of cytopathology at times after treatment when similarly treated

but infected cells show gross cytopathic effects.

I

E. EFFECT OF CONGO RED ON THE MORTALITY OF EGGS INFECTED WITH NDV

The results obtained are a remarkable vindication of the hypothesis

outlined in the introduction ( see review section). It must be concluded

from the results that the prevention of release of virus can bring about

the death of an embryo, if a sufficient number of cells are infected,

even if the virus strain is normally avirulent. This agrees with the

conclusion of Reeve et al (1970) that the accumulation of viral antigen,

which is presumably related to the rate of growth since they suggest

virus is released at the same rate, is directly related to virulence.

This is particularly supported by the speed with which high doses of

virulent virus in the presence of congo red reached 1007. mortality

(48-54 HR after infection) compared with high doses of avirulent

virus in the presence of congo red (96-168 HR).

169 F. THE SYNTHESIS OF NDV-INDUCED PROTEINS

Using the double labelling technique described, altogether six

proteins were seen common to all strains, if it is assumed that protein

'71' seen in gels from cells infected with Herts and Beaudette C

Corresponds to protein I seen in Queensland infected cells and pure

disrupted virus of all strains. Of the six protefns,A, I, II and III

correspond to polypeptides seen in pure virion preparations and can

be regarded as structural proteins. The major interest of these

proteins is the time at which they were produced after infection. The

virulent strains show all three major peaks HR after infection

while Queensland shows no obvious production until 3-4 HR. This is

further evidence that virulent strains are produced at a faster rate.

The two other polypeptide peaks NS„cand NS2 could not be identified

with structural proteins, NS2 was produced in very small amounts while

the production of NS1 even exceeded the production of the virus

proteins on one occasion. If NS1 and NS2 are the only two non-structural

proteins produced, at least in avirulent'infections, then one at

least would be expected to be an RNA-polymerise. NS1 was not detected

until the,6th hour which seems late for RNA-polymerase production

although experiments show that virus RNA production is not detectable

until 4-6 HR after infection (Results section H, also Brett and

Robinson, 1971). If NS1, NS2, or both, are responsible for RNA-

polymerase activity it is strange that production of these polypeptides

continue after the 'fall off' of RNA production. However the

apparent decline in RNA production seen in this study, and those of

Brett and Robinson (1967, 1971) with virulent strains may be due to

cytopathic effect:.

170 The significance of the induced polypeptides H1 and H2 which are

only detectable in Herts infections after the 10th hour, is difficult

to assess as they appear too late to have any relevance to the

differences in the virulence of the strains. One possibility is that

immediately prior to cell death, protein metabolism is sufficiently

disrupted to cause uptake of labelled amino acid into some cell

proteins even in the presence of actinomycin D.

The molecular weight of the RNA genome of NDV has been estimated

as approximately 6 x 10L6 daltons (Nakajima and Obara, 1967, Duesberg,

1968) which, using an average nucleotide molecular weight and an average

amino acid molecular weight, could code for protein of approximate

M.W. 800,000. The total estimated molecular weights of the four

structural proteins and the two non-structural NS1 and NS2 is 605,000

or 660,000 if the structural glycoprotein of molecular weight 56,000

identified by Mountcastle et al (1971) is included. This means that while

there is sufficient genetic information in the NOV genome to code for

both the structural and non-structural proteins detected in this study

it is unlikely that H1 and H2 are in fact coded for by the NDV genome.t,

It should be noted here that recent work (Huang, Baltimore and

Brett, 1971) has suggested that there is an RNA dependent RNA-polymerase

in the virions of NDV. This polymerase has such low activity that

it seems unlikely to be present in sufficient quantity to produce a

band on acrylamide gels, similarly early RNA production due to this

polymerase is unlikely to be detected by the method used in this study

to measure RNA production in cells. No evidence has been presented to

show that the virion polymerase is not a polymerase that is present in

cells infected with NDV late in infections and taken into the virion

particles with RNP at release. However if the virion polymerase is a

discrete protein it may account for polypeptides NS1 or NS2 or if

undetected by the double-labelling technique should have a molecular

weight not greater than 140,000.

171 Work with the effect of protein inhibitors on haemadsorption, cell

fusion and protein synthesis inhibition suggested a protein (or

proteins) necessary for these effects was produced in the first 4

hours after infection. Of the proteins induced by NDV only the structural

proteins and perhaps NS2 were detectable within 4 HR of infection.

172 G. PRODUCTION OF NDV-SPECIFIED RNA

The production of NDV RNA in cells offers further evidence that

the rate of formation of the virus products is important to the

virulence of the infecting strain. Most of the RNA produced, 80-90%

in Beaudette C infections, is in fact complementary to the parental,

strand (Kingsbury, 1970) so that production of RNA is not necessarily

a measure of virion production unless parental RNA production is directly

related to complementary RNA production.

Schafer, Pister and Schneider (1967) demonstrated the production

of viral antigens in the absence of RNA synthesis and proposed that the

NDV input strand of RNA, (+) RNA, acted as the sole messenger for virus

protein production. Reeve and co-workers (Reeve and Poste, 1971,

Reeve et al, 1971) have shown that even in the presence of azauridine,

which inhibits RNA synthesis, cell fusion and haemadsorption still

occur, findings that support those of Schafer et al (1967). Other

authors support the hypothesis that RNA complementary to the virus genome,

(-) RNA, acts as messenger RNA (Brett and Robinson, 1967, Blair and Robinson,

1968, Kingsbury, 1970) while Ralph (1969) has proposed that at

different stages of infection both (+) RNA and (-) RNA may act as

messenger RNA. in this present investigation increase in RNA production

due to NDV infection was not detected earlier than 4 HR after infection.

Structural proteins however were detected some time earlier than this,

1-2 HR after infection in the case of the virulent strains, which

supporta the hypothesis that, initially at least, the NDV genome

acts as messenger RNA. Of course RNA production before the 4 HR may

be merely too small tallye detected by the method used.

173 H. GENERAL DISCUSSION

Table 20 summarises the relationship of the various parts of this

work to virulence. Most of the phenomena that relate to virulence are

associated with the rate of production of virus. Cell fusion and

protein synthesis cut off appear to have no such relationship. The

theories on the cause of cell fusion have been previously discussed

but if massive cell modification by the virus could be responsible

then presumably this would relate to the rate of growth of the infecting

strain. Although protein synthesis inhibition by NDV and other viruses

has been thoroughly investigated little is known of the actual mechanisms

involved (Martin and Kerr, 1968) for this reason it is difficult to

relate protein synthesis inhibition in NDV to any other aspect of

virus infection.

The results obtained showing the effect of congo red on virulence,

in addition to the accumulation of neuraminidase and the other virus

antigens (Reeve et al, 1970), suggests that accumulation is responsible

for the death of NDV-infected cells and eggs. However this does not

explain why, if virus is produced at a faster rate,during infection by

virulent strains, it is not merely released at a faster rate and

accumulation prevented. Unless, of course, the release of virus is

controlled by the cell and not the virus. This may be so, Compans

et al (1966) showed that BHK 21-F cells infected with SV5 accumulated

viral RNP, released very little mature virus and were eventually killed,

while , primary rhesus monkey kidney (MK) cells, infected with the same

virus, released infective virus to high titres, did not accumulate

RNP and were not destroyed. Further to this, studies of NDV growth

rates have shown no differences in released virus titres that can be

related to virulence (Reeve and Waterson, 1970, Pennington, 1967,

Drain, 1969, Reeve et al, 1970.)

174 15

If the inference/that the rate of release of virus particles is

constant or approximately so, while the rate of productiOn varies

with strain,two states of infection could occur:

i) Infection in which the rate of viral production is less than

or equal'to the rate of virus release, i.e. the strictly

avirulent strains Queensland and Ulster.

ii) Infections in which the rate of production exceeds the rate

of release, causing accumulation of viral products and eventual

cell death. The virulent strains would be in this category

and the degree of virulence vary with the amount the rate

of production of viral antigens exceeded the rate of virus

release. So that virus production in Harts infections would

be far in excess of virus release while B1 (which forms

plaques occasionally and can kill eggs, see Table 1, also

Drain, 1969) would have a virus production rate only slightly

above the rate of release.

1 5 Table 20

Summary of the relationship to virulence of the properties investigated

Related to virulence

Not related to virulence

Accumulation of neuraminidase. Biophysical properties.

The extent of cell fusion

Biochemical properties of

of infected chick cells. neuraminidase.

The extent of haemadsorption Structural proteins.

to infected chick cells.

Inhibition of cellular Induced proteins up to 9 HR after

protein synthesis after infection. (After this time Herts

infection. infected cells induced two unique

protein; the relevance of which is

lleath of eggs after high discussed in the text).

doses of virus in the

presence of congo red.

Time of appearance of induced

proteins in infected chick cells.

ProductiOn of RNA in infected

chick cells treated with

Actinomycin D.

It has also been demonstrated that high doses of avirulent virus

show much higher lethality to eggs in the presence of congo red.

176

V. CONCLUSIONS

The relationship to virulence of the properties investigated are

summarised in Table 20. It is concluded from these results and previous

work by other authors:

1. Death of cells infected with NDV is a consequence of

accumulation of virus products in the cell.

2. Accumulation is related to the rate of production of the virus

components, therefore variations in rate cause variations in

virulence.

VI. SUGGESTIONS FOR FURTHER WORK

This work suggests that the rate of production of the virus components

in cells is directly related to the virulence of the infecting strain.

The most obvious step in the continuation of this work is therefore

to determine why production should be at a faster rate with one strain

than another. Since the cell's mechanism is used for the translation

of viral messenger-RNA it can be assumed that the limit to virus protein

production, which appears from this work to be responsible for cyto-

pathogenecity, is the production of viral messenger-RNA. Since RNA

production also appears to be related to virulence it would be significant

to study the NDV specified RNA-dependent RNA polymerase made during

NDV infections (Scholtissek and Rott, 1969) for differences between

strains with respect to both the rate of production by the polymerase

and the species of RNA produced.

In this study proteins produced during infections with either

virulent or avirulent strains have been shon to be similar. However

only extracts of whole cells have been examined, it is suggested that

sub-cellular fractions should be examined using similar techniques

to determine the exact site of virus specified protein production.

The hypothesis is that virulent strains may be able to modify membranes

177

that the avirulent strains cannot. Bang (1951) has shown that while

virulent strains appeared to develop within the cytoplasm avirulent

strains were formed only at the cell surface. The theory that lysosome

damage may be responsible for the initial cytopathic effect - cell

fusion, adds significance to such a study.

One of the more fundamentally important considerations in this

thesis is that the parental ANA may alone code for virus proteins.

Although Reeve and Poste .(1971) and Schafer et al (1967) both reached

the same conclusion using different techniques to measure viral

protein production in both cases the inhibitor 6-azauridine was used.

Similar experiments using a wide range of RNA inhibitors are now

necessary to confirm the results obtained with 6-azauridine. Double'

labelling experiments could be run in conjunction with experiments in

which RNA production is inhibited to determine whether all the virus-

induced proteins can be coded for by the input strand.

178

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Reprinted from MICROBIOS

I sopycnic separation of Newcastle disease virus on sucrose gradients using zonal ultracentrifuge rotors

Peter Reeve and Dennis J. Alexander

Department of Virology. Royal Postgraduate Medical School Hammersmith Hospital London W12 Great Britain

Abstract

H ighly purified preparations of Newcastle disease virus were obtained from allantoic fluids following centrifugation on sucrose or potassium tartrate gradients in an MSE BXIV zonal ultracentrifuge rotor.

Introduction

The use of zonal rotors for the purification of viruses is well established. For example, Fox and others have described the use of both continuous flow and batch type rotors for separating Semliki Forest virus and polyoma virus from cell culture fluids (Fox et al., 1967; Fox et al., i 968). Our studies on the properties of strains of Newcastle disease virus (NDV) required relatively large amounts of highly purified virus. This paper describes the isopycnic separation of NDV from allantoic fluids using both sucrose and potassium tartrate gradients; we also describe a simple discontinuous gradient method which should be applic-able to other systems.

Materials and methods

Viruses

The following strains of NDV were used • Herts 33 (Herts), received from W. H Allan, Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Weybridge, Surrey, England; isolated at Weybridge in 1933 by Dobson (1939). Italien, received from Professor R Rott, Institute of Virology, Giessen, Ger-many; isolated in Italy (Schafer, Schramm and Traub, 1949). Ulster, received from J. B. McFerran, Ministry of Agriculture for Ireland, Belfast, Northern Ireland; isolated in 1966 in Northern Ireland from the faeces of apparently healthy chickens.

Virus culture Batches of 200-500 ten-day-old fertile eggs were inoculated by the allantoic route with about to5 ° egg infectious doses (EID„) per embryo. Forty eight hr later the eggs were chilled for about 4 hr and the allantoic fluids harvested.

Virus (strain Herts 33) grown in BHK21 cells was obtained from Dr M. Berry, Glaxo Laboratories, Greenford, Middlesex, England. Virus containing 3H-uridine was obtained by inoculating each chick embryo with too µc uridine-5-T (purchased from the Radiochemical Centre, Amersham, Buckinghamshire, England) : acid-insoluble radioactivity was determined using a Packard 300 Tricarb Liquid Scintillation Spectrometer after precipitation onto cellulose acetate membrane filters with ice cold 5% (w/v) trichloracetic acid.

53 Microbios 1970 5 53-58

24cT

22C, A

E120

!so 0

4 8 12 16 20 24 36 03 36 40 44 46 50 58 GO 54

20

0 o 18- 0

le-

c 12-

Z 10—..

n 6-

126

1 24

1 22

1 20

116

1 16

1

0

110

1 08

Virus purification Virus from infected allantoic fluid or tissue culture medium was centrifuged at 40,000 x g for 3o min and resuspended to i / 1 oo original volume in phosphate buffered saline (PBS) at pH 7.4 before centrifuging in a sucrose or potassium tartrate gradient in an MSE BXIV titanium zonal rotor at 40,000 rpm

00,000 x g) using an MSE 65 ultracentrifuge. Fractions were collected using an LKB Ultrarac fraction collector after measuring ultraviolet absorption at 259 nm in an LKB Uvicord II. When material, e.g. radioactive virus, was scarce, samples were centrifuged at 30,000 rpm ( too,000 x g) in sucrose or potassium tartrate gradients in 23 ml tubes using a swing-out rotor.

Sucrose and potassium tartrate solutions (w/w) containing (poi M tris pH 7.4, were made using the tables given in the Handbook of Chemistry and Physics (Weast, 1968) : densities were estimated from the refractive index of the solutions using the same tables.

Protein concentrations Protein concentrations were estimated by Lowry's method (1951) using crystal-line bovine serum albumin as a standard. Haemagglutinin was titrated using t•o% chicken erythrocytes in o • 5 M NaCl. Tests were done in plastic trays and the last dilution giving complete agglutination was taken as the end point.

Results

When virus was centrifuged for 6o—go min at 100,0w X g on continuous gradients made using an MSE gradient mixing device two major peaks absorb-ing ultraviolet light were seen. The viral haemagglutinin was distributed throughout the gradient, but a major peak could be seen, as well as two smaller peaks at lesser densities (Figure 1). Similar results were obtained with gradients made using io% to 6o% (w/w) sucrose or i ocy„ to 40% (w/w) potassium tartrate. The result was the same whether centrifugation continued for 6o min,

Fraction number in ml each)

Figure 1 Purification of NOV strain Italien on a sucrose gradient BkIV titanium rotor centrifuged 40,000 rpm for 90 min protein N—N solution density Histogram reciprocal of HA titre

54 Microbios P Reeve and D J Alexander

8 12 II 20 24 28 32 36 40

Den

sit

y 9

/m

1

1 22

120

118

ire

114

1 12

110

0 0

a 0.

2 4 6 8 10 12

Fraction number

Figure 2 Purification of NDV, strain Italien on a sucrose gradient Twenty three nil tubes in a swing out rotor 30 000 rpm 90 min 0-0 solution density .--110 infectivity PFU per ml III—IIII 3 H radioactivity Histogram reciprocal of HA titre

go min or 18 hr. Examination of material sedimented by subsequent high speed centrifugation from the major haemagglutinin peaks found at densities of about i •o4; I • I 2 and 1 • 18 g per ml using a Philips EM 30o electron microscope re-vealed typical intact virus particles at the highest density but only fragments at lower densities. Gradient centrifugation performed using tubes and a swing-out rotor showed that virus infectivity and radioactivity of 3H-labelled virus was associated with the peak of highest density (Figure 2).

In order to determine whether virus preparations were originally heterogene-ous or had been degraded during purification, material from fractions corres-ponding to the three major haemagglutinin peaks was sedimented and recentri-fuged in sucrose or potassium tartrate gradients. Single peaks were obtained at the same densities as those from which the material had originally been obtained, suggesting that the purification procedure had not degraded previously intact particles (Figure 3).

Fraction number (to mt each)

Figure 3 Centrifugation of NDV purified by zonal centrifugation on sucrose gradient BXIV titanium rotor, 40 000 rpm for 90 min iiii—ip solution density Histogram reciprocal of HA titre

55 Zonal centrifugation of NDV

0

O O •— 30

E 20

O

160 ,3,

40

4 6 12 16 20 24 26 32 36 40 44 4

1 22

1 20

1 16

1 16

1 14

1 12 E 0

1 10

Discontinuous gradients

Discontinuous gradients were made to obtain a sharper separation of intact NDV particles from host material and non-infective haemagglutinating frag-ments. Gradients were made by filling the zonal rotors with three separate sucrose or potassium tartrate solutions with densities of about 1.04, 1-16 and I •26 g per ml. Using sucrose, gradients were obtained with two 'steps' at which the density changed rapidly. After centrifugation for go min at 4°C, virus banded on such gradients to give two or three sharp peaks of haemagglutinin. Infectivity was associated with a major peak isopycnic with sucrose at densities of about i •i8 g per ml and this was easily separated from other fractions which only contained haemagglutinating virus fragments (Figure 4). If centrifugation was continued for 18 hr, or gradients were allowed to equilibrate by centrifuging at 2500 rpm for 1-2 hr before use, more mixing occurred between the sucrose solutions and less sharp changes of density in the subsequent gradients were seen. With potassium tartrate solution discontinuous gradients could not be obtained: evidently the lack of viscosity of potassium tartrate permitted ready mixing so that almost linear gradients were obtained.

Fraction number ( lornl each)

Figure 4 Purification of NDV on a discontinuous sucrose gradient— BXIV titanium rotor 40 000 rpm for 00 min 0-0 solution density Histogram reciprocal of HA tine

Estimation of virus purity

The protein concentration of the fractions was compared with the titres of haemagglutinin Estimates of purity were also obtained by subjecting virus sus-pension to electrophoresis on 7-5% polyacrylamide gels in a Shandon disc electrophoresis apparatus run at a constant current of I or 4 mA. Infected allantoic fluid contained about 1-2 mg protein per ml and as many as 5 bands were seen on polyacrylamide gels stained with 1% amido black in 7% acetic acid (Table 1, Figure 5a).

Approximately go% non-viral protein was removed after sedimenting virus from infected fluid but such virus still produced several bands on polyacrylamide


Table 1 Purification of NDV, strain Italien, from allantoic fluid. After centrifuging at 20,000 rpm for 30 min to sediment virus the concentrate obtained was centrifuged on a sucrose gradient in a BXIV zonal rotor for 90 min at 45,000 rpm.

Haemagglutinin per ml

Protein Ag per ml

Haemagglutinin per mg protein

Original allantoic fluid 5,120 2,000 2,560

Concentrate from allantoic fluid 163,840 2,600 63,000

Virus after zonal centrifugation 40,960 50 819,200

gels indicating the presence of several contaminating soluble proteins (Figure 5c), but after zonal centrifugation virus suspensions were obtained containing about ro5 HNU per mg protein which did not produce adventitious bands following polyacrylamide gel electrophoresis (Table t, Figure 5e). Tests showed that less than zpo eg bovine serum albumin could be detected by polyacrylamide gel electrophoresis so that virus suspensions purified by zonal centrifugation were substantially freed of contaminating soluble host proteins, but of course, host protein may well have remained closely adsorbed to virus particles.

Figure 5 Polyacrylamide gel electrophoresis of NDV infected allantoic fluid. About 200 Ag protein was layered onto 7.5% polyacrylamide gels before electrophoresis at 4 mA pH 9.2. (a) Original infected allantoic fluid. (b) Allantoic fluid after low speed centrifugation. (c) Pellet obtained after high speed centrifugation. (d) Supernatant obtained after high speed centrifugation. (e) Virus suspension after zonal centrifugation.

Discussion

Satisfactory isopycnic separation of NDV from infected allantoic fluids was obtained using either sucrose or potassium tartrate gradients. Duesberg and Robinson (1967) stated that `NDV could not be banded on sucrose' since sufficiently high densities could not be obtained. However, using solutions made on a weight for weight basis, as given in the Handbook of Chemistry and Physics ( 1968), sucrose solutions can readily be obtained with densities of up to 1.28 g per ml and this is sufficiently high to permit isopycnic banding of NDV, whose mean density is about i •18. Sucrose permitted discontinuous gradients to be made without the use of a gradient mixing machine. With such gradients it was possible to separate readily complete infectious NDV virions from the heterogeneous suspension obtained from infected allantoic fluids. Virus grown in

57 Zonal centrifugation of NDV

cell culture proved difficult to purify, because large amounts of contaminating host protein always remained associated with virus suspensions. No differences were seen in the behaviour of the different strains and there were no differences in the mean densities of the purified virus even though these strains exhibit great differences in virulence for chick embryos and cell cultures (Waterson, Penning-ton and Allan, 1967). Further, no morphological differences were seen when negatively stained preparations of the isolated virions were examined in the electron microscope.

Acknowledgements

We thank Mr J. Hall and Miss Joy Pacey for excellent technical assistance, Mrs J. D. Almeida for electron microscopy and Professor A P Waterson for his encouragement and help with the manuscript This work was aided by grants from the Wellcome Trust and the Agricultural Research Council

References

Fox S. M , Birnie G D., Martin E M. and Sonnabend J A. 1967 Isolation of polyoma virus and Semliki Forest virus from tissue culture fluid by continuous flow zonal ultracentrifugation J gem Virol 1 577

Fox S M , Birnie G D , Harvey D R.. Martin E M. and Sonnabend J A. 1968 The use of batch-type zonal ultracentrifuge rotors for the isolation and purification of viruses J gen Viral 2 455

Dobson N 1939 Newcastle disease Proc 7th World s Poultry Cong Cleveland. 250

Lowry 0. H , Rosebrough N. J , Farr A L and Randall R. J 1951 Protein estimation with the Folin phenol reagent J biol Chem 193 265

Schafer W , Schramm G and Traub E 1949 Untersuchungen uber das Virus der atypischen Geflugelpest Z Naturforschung 46157

Weast R C 1968 Editor-in-Chief of The Handbook of Chemistry and Physics 4th edition 1967-8 The Chemical Rubber Company Cleveland Ohio U S A

Accepted 2 December 1969


THE FACULTY PRESS 88 Regent Street Cambridge England

Reprinted from MICROBIOS

Characterization and biological properties of the neuraminidase of strains of Newcastle disease virus which differ in virulence

Dennis J Alexander, Peter Reeve and 'W. H Allan

Department of Virology Royal Postgraduate Medical School London W 12. Great Britain and 'Ministry of Agriculture Fisheries and Food Central Veterinary Laboratories New Haw Weybridge Surrey, Great Britain

Abstract

Different strains of NDV vary widely in virulence and cytopathogenicity The biological and biochemical characteristics of the virus-bound neuraminidase of a representative number of NDV strains were examined All the strains were found to have essentially identical neuraminidase in vitro However, the levels of cell-associated neuraminidase in the chono-allantoic membrane 22 hrafterinfection appeared to be related to thevirulenceof the infecting strain.

Introduction

The enzyme neuraminidase (mucopolysaccharidc N-acetylneuraminyl hydro-lase EC 3 2.1. I 8) has been found to be distributed among many tissues, micro-organisms and, in particular, the myxoviruses In general the study of myxo-virus neuraminidase has been concerned mainly with influenza virus, and the characteristics, structure and morphology of influenza virus neuraminidase have been extensively studied (Rafelson, Schneir and Wilson, 1963; Drzeniek, Seto and Rott, 1966; Drzeniek, Frank and Rott, 1968, Kendal, Biddle and Belyavin, 1968; Paniker, 1968).

Different strains of Newcastle disease virus (NDV) show a continuous spectrum of differing virulence, as measured by lethality in chickens or eggs (Waterson, Pennington and Allan, 1967), and cytopathogenicity (Schloer and Hanson, 1968). This system is therefore particularly useful in studies of the relationship between viral structure and activity and virulence or cytopatho-genicity.

Waterson et al. (1967) have suggested that difference in virulence of the different strains of NDV could in some way be related to the neuraminidase activity. This suggestion was given more weight by the work of Scholtissek, Becht and Drzeniek (1967), who showed that cytopathogenicity of influenza virus was related to neuraminidase activity. Furthermore it has been shown by Kohn and Fuchs (1969) that other properties of the NDV virion are not re-lated to virulence.

Drzeniek et al. (1966), in a preliminary study, characterized the neuramini-dase, both virus-bound and released, of two strains of NDV (Italien and Beau-dette C) and showed that they were alike in their properties. In this work we have limited our studies to virus-bound neuraminidase but have examined a larger number of strains of much greater variation in virulence and cyto-pathogenicity, and included other biological properties of the NDV neura-minidase.

155 Microbios 1970 6 155-165

Materials and methods

Viruses

The following strains of NDV were used: Field Pheasant — isolated at the Central Veterinary Laboratories, Weybridge, Great Britain, 1962. Herts 33 (Herts) — Dobson (1939). Italien — Schafer, Schramm and Traub (1949). Texas G.B. (Texas) — isolated near Austin, Texas, U.S.A., 1948. Warwick — isolated in Great Britain, 1966. Eastwood Notts — isolated in Great Britain, 1966. Queensland V4 (Queensland) — isolated in Brisbane, Australia, 1966. Ulster — received from J. B. McFerran, Ministry of Agriculture for Northern Ireland, Belfast, and isolated in 1966. B1 — Hitchner and Johnson (1948) . F — Asplin (1952). Bcaudette C — Granoff (1959). Virus was grown in the allantois of io-day-old fertile hen's eggs. The allantoic fluid was harvested, centrifuged at low speed to remove cell debris, and the virus sedimented at 45,000 x g for 45 min. The virus was partially purified (> 95% of protein removed) by sedimentation through 32% (w/w) sucrose onto a 6o% (w/w) sucrose cushion. In experiments involving haemagglutinin and infectivity titrations, infected allantoic fluid was used without further treatment.

Measurement of virulence and ability to form plaques

The methods used for the estimation of intracerebral pathogenicity index (ICPI) in chickens, and mean death time in eggs (MDT) have been described in detail before (Methodsfor the examination of poultry biologics, 1963).

Primary chick fibroblast monolayers, overlaid with 0.9% (w/v) nutrient agar and incubated at 37°C, were examined for plaques 3 days after infection with virus.

Titrations of haemagglutinating activity and infectivity

Haemagglutinin (HA) was measured in standard plastic plates in 0•2 ml

volumes, phosphate buffered saline pH 7.2 (PBS) was used as diluent with I% (w/v) chicken red blood cells.

Infectivity for chick embryos was estimated by inoculating o•i ml volumes of serially diluted virus suspensions into the allantoic cavity of 9- or o-day-old fertile chicken eggs. Eggs were candled daily and after 7 days the allantoic fluid of surviving eggs was tested for the presence of hacmagglutinin The 5o% egg infective dose (EID„) per ml was estimated by the Kaerber method.

Neuraminidase assay

Neurammidase activity was estimated by measuring the amount of N-acetyl neuraminic acid (NANA) released from a suitable substrate The solution to be tested (0.1 ml) was incubated with o•1 ml substrate and o.3 ml of buffer for io min at 37°C. The NANA released was estimated by the method of Aminoff

156 Microbios D J. Alexander, P Reeve and W H Allan

(1961). The substrate used was either bovine neuramme lactose (Calbiochem Ltd) 200 jig per test, or the equivalent amount of fetuin prepared from foetal calf serum (Graham, 1961).

Enzyme activity was expressed as jig of NANA released.

Release of neuraminidase activity by Tween 20 at pH 10.0

Virus was sedimented at 45,00o x g for 45 min and resuspended in 1% v/v Tween 20 in bicarbonate buffer pH to•o (Webster and Darlington, 1969) and incubated at 37°C overnight. The mixture was then centrifuged at 45,000 x g for 6o min, the pellet resuspended to volume and both suspensions tested for neuraminidase activity In each case a control was performed under identical conditions but was incubated in bicarbonate buffer which did not contain Tween 20.

Estimation of cell-associated neuraminidase activity

For each strain tested i x 109 EID„ were inoculated into the allantoic cavity of o-day-old eggs. After 22 hr incubation at 37°C the chorioallantoic membrane

was removed from each egg, washed three times in ice cold PBS and stored at — 7o°C until assayed. The membranes were thawed and blended in homogeni-zers with motor driven Teflon pestles in sodium acetate buffer (pH 5.2). The homogenate was then assayed for neuraminidase activity and protein was estimated by the method of Lowry et al. (1951).

Results

Virulence and cytopathogenicity

The strains used covered a wide range of virulence, whether measured by intracerebral pathogenicity index in chickens or mean death time in eggs

Table 1 Virulence and cytopathogenicity of different NIDV strains

Strain ICPI MDT Plaguing ability

Field Pheasant 2 00 54 + + Herts 1 88 49 + +

Italian 1 86 50 + + Texas 1 75 55 + + Warwick 1 72 62 + + Eastwood Notts 1 70 70 Beaudette C 1 46 62 + B, 040 117 ± F 0 25 119 — Queensland 0 16 ix, — Ulster 0 00 ci, —

+ + Large well-defined plaques + Small well-defined plaques ± Plaques very small, ill defined and often absent — Does not form plaques ICPI Intracerebral pathogenicity index MDT Mean death time in eggs in hours

1 57 N DV virus-bound neuraminidase

(Table 1). The two methods of estimation generally run inversely parallel, Beaudette C and Field Pheasant being exceptions.

Ability to form plaques in chicken embryo cell sheets, although only the gross differences were measured, varied with virulence.

Relationship of neuraminidase to HA and infectivity

In order to determine if any strains possessed more neuraminidase than others the neuraminidase activity was compared with HA titre (Table 2a). HA was taken to be a measure of viral surface area and preferred to a particle count because of the pleomorphism of the virus. The results show that the ratio of neuraminidase activity to HA units is generally constant, except for strains Beaudette C and, to a lesser extent, Ulster which have considerably lower ratios.

Table 2a Relationship between haemagglutinin and neuraminidase activity

Strain Neuramimdase activity/m1 HA/ml

Neuraminidase HA x 10-3

Field Pheasant 38 0 1500 25 4

Hurts 360 0 10240 35 0

Texas 87 5 2560 34 1

Warwick 84 5 2560 33 0

Eastwood Notts 171 0 5120 33 3

Beaudette C 271 0 20480 13 3

B, 176 0 5120 34 4

F 74 5 2560 28 1

Queensland 172 5 5120 33 6

Ulster 845 0 40960 20 6

The relationship between HA, neuraminidase and infectivity was also investigated. The HA/EID50 and the neuraminidase/EID„ ratios, again with the exceptions of Beaudette C and Ulster, even after allowing for their high HA titres, were very similar for all strains (Table 2b).

Table 2b Relationship between infectivity, haemagglutinin and neuraminidase activity

Strain HA Neuraminidase activity (pH 6 0)

EID„ x 10-7 ElDso x 10-9

Harts 2 9 85

Texas 39 73

Eastwood Notts 36 75

Beaudette C 196 22 2

24 52

F 23 103

Ulster 101 142

158 Microbios D J Alexander, P Reeve and W. H. Allan

0 12

42 0 04 z a z

Kinetic studies of virus-bound neuraminidase

The amount of NANA released after to min was plotted against the concentra-tion of substrate used. All eight strains examined gave kinetic curves essentially Identical to that of strain Herts (Figure 1 ). The Michaelis-Menten constants

0 10

0 20

0 30 0 40

Neuramine lactose [}moles )

Figure 1 Kinetics of the virus-bound neuraminidase of strain Herts Standard enzyme assay procedure was used except that the concentration of neuramine lactose was as above, pH was 5 4 with 0 1 M sodium acetate-acetic acid buffer

(Km) were obtained by Lineweaver-Burk plots. All the strains had a Km of approximately i x io-3 m (Table 3).

With fetuin as substrate the optimum pH of the neuraminidase of the different strains, though varying slightly from experiment to experiment, always fell in the range pH 5-1-5.3 (Table 4) and was considered to be the same for all the strains tested. Typical curves of pH effect with fetuin as a substrate are shown in Figures 2a and 2c. A second minor peak at pH 6.3 was seen with all the strains tested except strain F. If neuramine lactose was used as the substrate the pH

Table 3 Michaelis- Menton constant of neuramindase at pH 5 4

Strain Km

Field Pheasant 1 0.10-3M Harts 10x10-3M Italien 0 9.10-3M Texas 1 0 x 10-3M Beaudette C 1 0.10-3M

F 09x10-'M Queensland 0 9.10-3M Ulster 1 0.10-3M

159 N DV virus-bound neuraminidase

12

a

O

12

Neura

mmid

ase

a

4

0

Table4 The effect of pH on neuraminidase activity using fetuin as a substrate

%of optimum pH of minor neuraminidase

Strain pH optimum peak of activity activity at pH 7 2

Harts 5 1-5 2 63 534 Texas 5 1-5 2 6 3 60 0 Beaudette C 5 2-5 3 6 1 69 0 F 5 2-5 3 No minor peak 62 5

Ulster 5 1-5 2 6 3 49 7

A B

- ..,, A----

- - 1 \.

5 7 a a r

p H

Figure 2 The effect of pH on virus-bound neuraminidase under different conditions The normal enzyme assay procedure was used except for the pH. buffer and substrate which were A — Herts strain, buffer for pH 4 2-5 5, 0 1 M sodium acetate-acetic acid buffer pH 5 6-7 2 0 1 M phosphate buffer, substrate tot= B — Herts strain, buffer acetate/phosphate , substrate neuramine lactose C — Ulster strain, buffer L—A acetate/phosphate .0-0 0 2 M sodium acid maleate buffer, substrate fetu in D — Herts strain, buffer sodium acid maleate , substrate neuramine lactose

160 Microbios D J Alexander, P. Reeve and W. H. Allan

s-?

Neura

min

ida

se

0

0

S

S

100

60

4

6

20

optimum for all the strains was raised slightly to pH 5.4 (Figure 2b), the second minor peak was far more obvious although it still occurred at pH 6.3. In these experiments sodium acetate buffer was usually used for pH values from 4.1 to 5.5, phosphate buffer for pH 5.6-7'2. If sodium acid maleate buffer was used from pH 5.1 to pH 7.2 with both fetuin and neuramine lactose the two peaks were present but with neuramine lactose the 'minor' peak was so much greater that it became the larger of the two (Figures 2c and 2d).

In Table 4 the neuraminidase activity at physiological pH (pH 7.2) is shown as a percentage of the activity at the optimum pH. The strains tested were generally in the range of 50-7o% and although differences in the strains were consistently found there was no relationship with virulence

Heat stability of virus-bound neuraminidase

The heat stability of virus-bound neuraminidase was estimated by incubating virus suspensions at 56°C and measuring the remaining neuraminidase activity at intervals. Within 20 min the neuraminidase activity had been destroyed in all the strains tested except Beaudette C, which still had 3o% of the original activity (Figure 3).

0

5 10 15

20

Exposure time I miri I

Figure 3 Heat stability of virus-bound neuraminidase of different strains of NOV at 56°C At specified times 0 1 ml of enzyme was added to 0 3 ml of ice cold buffer to stop further heat inactivation . 0-0 Herts: III—E Texas, A—A Beaudette C .E1-0 Queensland ,0-0 Ulster.


Table 5 Effect of Tween 20 (1% v/v pH 10 0) on virus-bound neuraminidase

Strain Percentage activity of total control activity Control Treated

Field Pheasant Supernatant 10 0 125 0 Pellet 90 0 13 3

Hens Supernatant 3 0 16 7

Pellet 97 0 0 0

Texas Supernatant 4 1 79 6

Pellet 95 9 8 5

Beaudette C Supernatant 10 0 20 0

Pellet 90 0 2 2

Queensland Supernatant 8 3 92 5

Pellet 91 7 12 1

Ulster Supernatant 1 7 70 0

Pellet 98 3 7 6

'Control' samples were incubated overnight at 37°C with bicarbonate buffer pH 10 0 For 'treated' samples the buffer contained 1% v/v Tween 20 All samples were spun at 45,000 x g for 60 min, the pellets resuspended to volume and the solutions tested for neuraminidase activity

Effect of Tneen 20 at pH 10.0

Treatment of virus with 1 % v/v Tween 20 at pH to-o caused the release of neuraminidase with all the strains tested; between 7o% and t00% of the re-maining activity was present as free neuraminidase that could not be sedimented (Table 5). The total yield after treatment varied considerably from strain to strain. With strains Herts and Beaudette C there was an 8o% loss of neuramini-dase activity while with Field Pheasant there was an increase of nearly 40%.

Table 6 Cell associated neuraminidase levels in infected chorioallantoic membranes 22 hr after infection

Strain Neuraminidase activity/mg protein

MDT (hours)

Harts 11 1 49

Texas 7 3 55

Warwick 5 9 62

Beaudette C 4 6 62

Eastwood Notts 2 8 70

B, 1 5 117

F 1 6 119 Queensland 0 8 m

Ulster 0 5 co

162 Microbios D J Alexander, P. Reeve and W. H Allan

Neuraminidase activity in infected chorioallantoic membranes

The levels of neuraminidase per mg protein in the chorioallantoic membranes of eggs infected with different strains appear to be related to the virulence of the infecting strain (Table 6). The plaque-forming strains, i.e. Eastwood Notts and the more virulent strains, show a linear relationship between cell associated neuraminidase and MDT. The less virulent strains show less marked differ-ences in neuraminidase levels, presumably because there is very little accumula-tion of the enzyme and much of the activity recorded is due to whole virus particles associated with the membrane.

Discussion

None of the properties of NDV neuraminidase examined show any great variation from strain to strain. Unlike influenza virus (Rafelson et al., 1963) it appears that different NDV strains possess neuraminidases which are essentially identical in their biochemical properties.

On assessment of physical characteristics Beaudette C appears to be the most atypical strain. The heat resistance shown by Beaudette C neuraminidase is probably due to the protection of the intact virion since Drzeniek et al. (1966) have shown that neuraminidase isolated from Beaudette C virus is just as heat labile as neuraminidase from the other strains. Beaudette C has twice as much hacmagglutinin as other strains which could account for the protection of the neuraminidase. Rott, Reda and Schafer (1962) have shown that a large fraction of the virus in their Beaudette C suspensions was devoid of ribonucleo-protein. From the HA/EID„ ratios which arc, in fact, estimation of non-infectious particles this does not appear to be a general characteristic of all the strains although Ulster also seems to have a high HA/EID5O ratio.

It is difficult to assess the significance of the differences in the effect of Tween 20 on the neuraminidase of the different strains. Presumably the enzyme of those strains showing a low yield of released neuraminidase after Tween 20 treatment is more intimately associated with the lipid membrane of the virus, or that their enzyme is different in some way and rendered inactive in the presence of Tween 20.

The pH optimum of NDV neuraminidase is lower than that recorded by Drzeniek et al. (1966) for Italien and Beaudette C. However, this is probably due to variation in the substrate. It has been shown by Raffelson et al. (1963) that pH optima of neuraminidase differ with substrate. The observation of two peaks of enzyme activity has been previously recorded by Drzeniek, Bogel and Rott (1967) with the neuraminidase of bovine parainfluenza 3 virus. Since in both the substrates we have used, the NANA is not specifically attached the two peaks could be produced by the availability of substrate varying with pH. However, Drzeniek (1967) using 2-3' and 2-6' linked sialolactose, has shown that NDV neuraminidase, unlike Vibrio cholerae neuraminidase cleaves only 2-3' linkages and that the failure to cleave 2-6' linkages is unaffected by pH changes. Alternatively the enzyme may exist in more than one form, with different pH optima.

163 NDV virus-bound neuraminidase

The relationship between virulence and cell-associated neuraminidase levels has been noted previously by Reeve, Rosenblum and Alexander (1970) who have shown that all the major virus antigens accumulate in the chorioallantoic membrane of eggs infected with virulent strains but not in eggs infected with avirulent strains of NDV. Accumulation of neuraminidase and other viral antigens in virulent strains infers that there will be a far more extensive modifi-cation of the cell membrane than with avirulent strains and this alone could cause cell death. However, it is also possible that cell death could be brought about by generalized disruption of cell membranes caused by the accumulating neuraminidase, or by the action of neuraminidase at a specific site or sites in the cell (Bang, 1953; Allison and Mallucci, 1965).

Acknowledgements

D J Alexander is supported by the Agricultural Research Council to whom we are in-debted for a grant for technical assistance and supplies This research was aided by grants from the Wellcome Trust and Action for the Crippled Child (Polio Research Fund) We are most grateful to Professor A P Waterson for his help and encouragement

References

Allison A. C. and Mallucci L. 1965 Histochemical studies of lysosomes and lysosomal enzymes in virus infected cell cultures J. exp med.121 463

Aminoff D 1961 Methods for the quantitative estimation of NANA and their application to hydrolysates of sialomucoids Biochem J 81 384

Asplin F. D 1952 Immunisation against Newcastle disease with a virus of low virulence (strain F) and observations on sub-clinical infection in partially resistant fowls Vet Roc, 64 245

Bang F. B. 1953 The development of Newcastle disease virus in cells of the chorioallantoic membrane as studied by thin section Bull Johns Hopkins Hosp 92 309

Dobson N.1939 Newcastle Disease Proc 7th Worlds Poultry Cong Cleveland p 250

Drzeniek R 1967 Differences in splitting capacity of virus and V cholerae neurammidase on sialic acid type substrates Blochem Blophys Res Comm 26 631

Drzeniek R , Bogel K and Rott R 1967 On the classification of bovine parainfluenza 3 virus Virology31 725

Drzeniek R , Frank H and Rott R 1968 Electron microscopy of purified influenza neuraminidase Virology 36 703 Drzeniek R , Seto J T and Rott R 1966 Characterisation of neuraminidases from myxoviruses Biochem biophys Acta 128 547

Graham E R. B. 1961 Some aspects of the carbohydrate moiety of fetuin Australian J So 24 140

Granoff A 1959 Studies on mixed infection with Newcastle disease virus Virology9 636

Hitchner S. B and Johnson E. P. 1948 A virus of low virulence for immunizing fowls against Newcastle disease (avian pneumoencephalitis) Vet Med 43 525

Kendal A P , Biddle F. and Belyavin G 1968 Influenza virus neuraminidase and the viral surface Biochem blophys Acta 165 419

Kohn A and Fuchs P 1969 Cell fusion by various strains of NOV and their virulence J Virol 3 539

Lowry 0. H., Rosebrough N J , Farr A. L and Randall R J 1951 Protein measurement with the Foil n phenol reagent J .61°1 Chem 193 265

Methods for the examination of poultry biologics 1963 Publication No 1038 National Academy of Sciences— National Research Council, Washington DC USA

Paniker C K. J 1968 Serological relationships between the neuraminidases of influenza viruses J gen Virol 2 385 Rafelson M E , Schneir M , Wilson V 1963 Studies on the neuraminidase of influenza virus II Additional properties of the enzyme from the Asian and PR8 strain Arch. Biochem Biophys 103 424

Reeve P , Rosenblum M and Alexander D J 1970 Growth in chorioallantoic membranes of strains of Newcastle disease virus of differing virulence J Hyg 68 61

Rott R , Reda I M. and Schafer W 1 962 Isolation and characterisation of haemagglutinating non infectious particles produced during multiplication of Newcastle disease virus (NDV) Virology16 207

164 Microbios D. J Alexander, P. Reeve and W. H. Allan

Schafer W.. Schramm G. and Traub E. 1949 Untersuchungen uber das Virus der atypischen Geflugelpest Z Naturforsch 46 157

Schloer G. and Hanson R. P. 1968 Relationship of plaque size and virulence for chickens of 14 representative Newcastle disease virus strains J.14rol 2 40

Scholtissek C , Becht H. and Drzeniek R. 1967 Biochemical studies on the cytopathic effect of influenza virus J.gen.Virol 1 219. Waterson A. P , Pennington T. H and Allan W. H. 1967 Virulence in Newcastle disease virus A preliminary study Br.med.Bul1,23 138

Webster R. G and Darlington R W 1969 Disruption of myxoviruses with Tween 20 and isolation of biologically active haemagglutinin and neurammidase subunits J Oro, 4 182

Accepted 7 April 1970


THE FACULTY PRESS 88 Regent Street Cambridge England .

Reprinted from CYTOBIOS

Plaque formation, cell fusion and haemadsorption by Newcastle disease virus

Peter Reeve and Dennis J. Alexander

Department of Virology. Royal Postgraduate Medical School Hammersmith Hospital London W12, Great Britain

Abstract

Only those strains of Newcastle disease virus (NDV) which Induced syncytia in chicic embryo fibroblasts formed plaques. Haemadsorption also was greater with plaque forming strains than with other strains, suggesting a more extensive modification of the cell surface.

The myxoviruses, which include Newcastle disease virus (NDV), are assembled at the cell surface, which they modify in such a way that erythrocytes can be adsorbed by the cells (haemadsorption). Cells infected with NDV are also known to fuse to form syncytia. We have examined strains of NDV grown in chick embryo fibroblasts to test the hypothesis that plaque formation depends on polykaryocytosis, which is itself determined by the extent of host cell modifica-tion. Virus pools were prepared from NDV-infected allantoic fluid of to-day chick embryos and stored at — 70°C until required.

The following strains were used, Herts 33 (Dobson, 1939), Texas G. B., Warwick, Beaudette C (Granoff, 1959), 'F' (Asplin, 1952), Queensland and Ulster. Of these the first four produced plaques in chick embryo fibroblast monolayers, whereas the other three did not. Although strains Queensland and Ulster failed to form plaques they grew well in chick embryo fibroblasts, re-leasing high titres of infectious particles (Reeve and Waterson, 197o). Poly-karyocytosis was looked for in monolayers infected with a multiplicity of about o egg infectious doses (EID„) per cell, incubated at 37°C and stained with

Giemsa 12 hr and 20 hr respectively after infection. Syncytia were seen after in-fection with strains Herts, Texas G.B., Warwick and Beaudette C (Table t). A few small syncytia were seen following infection with strain `F but these were absent in cultures infected with Queensland or Ulster. Syncytia varied in size, as judged by the number of nuclei seen 12 hr after infection; Herts and Texas G.B. inducing the largest and Beaudette C the smallest (Table ). Haemadsorption was estimated by adding i % chicken erythrocytes in phos-phate buffered saline (PBS) at pH 7.2 to cultures washed three times with o•15M NaCl. After 15 min at +4°C unadsorbed erythrocytes were removed by three further washes with saline. Cultures were then stained with Giemsa and the number of erythrocytes adsorbed to individual cells counted. Uninfected cultures failed to adsorb erythrocytes. With strains Herts, Texas G B., Warwick and Beaudette C, haemadsorption was seen 4-6 hr after infection, the number of erythrocytes adsorbed to individual cells increasing exponentially until about 12 hr after infection, when infected cells were covered with erythrocytes, syn-cytia began to appear and accurate counts were difficult. With strains `F', Queensland and Ulster, haemadsorption was always less than with the virulent

55 Cytobios 1970 5 55-57

a 10

9 6 5

3

Num

ber o

f ery

thro

cyte

s

Table 1 Comparison of the ability to form plaques and syncytia in chick embryo fibroblast cell cultures infected with strains of NDV. Number of nuclei per syncytium estimated from counts on at least 50 syncytia, 12 hr after infection

Strain Ability to form plaques Presence of syncytia Average number of nuclei per syncytium

Herts 33 + + 16 Texas GB + + 15 Warwick + + 10 Beaudette C + + 8

Queensland

—

— , 4

0 Ulster — — 0

strains, only about 8 erythrocytes adsorbed to individual cells 36 hr after in-fection (Figure t) ; occasionally cells or syncytia which had adsorbed larger numhers of erythrocytes were seen

Our strains show a direct relationship between plaque formation and the presence of syncytia, and it is known that the size of the plaques induced by strains of NM' varies, being directly related to their virulence for chick embryos (Schloer and Hanson, 1968). Our results suggest differences in the size of syncytia may determine plaque size. Roizman (1962) has shown that alteration of the cell surface is a prerequisite for polykaryocytosis, and described variants of herpes simplex virus which showed a correlation between plaque size and ability to form syncytia. We suggest that modification of the cell surface beyond a certain point induces cell fusion; with virulent strains this point is reached rapidly in all cells infected; with avirulent strains this point is rarely or never

fibro

bla

st

30 • ,

20 / • •

7' - /A

F 71 •

o - • I • %• 2. ❑ ii o

• II" , 0 • 0

1 o

0 1 /b I

2 °

4 8 12 16 20 24 28 32 36

Hr after inoculation

Figure 1 Haemadsorption by chick embryo fibroblasts infected with strains of N DV The figures given are mean values for 2-3 experiments for each strain, at least 50 infected cells were observed for each point • Herts 33 • Texas G B A Warwick VBeaudette C 0 F' ❑ Queensland L Ulster

56 Cytobios P. Reeve and D J Alexander

reached. The presence of haemagglutinin at the cell surface may by itself cause sufficient alteration to induce cell fusion. HoN‘ever, it is equally likely that this may only be a secondary phenomenon occurring in parallel with other cellular changes which are responsible for cell fusion. Poste (1969) has suggested that cellular lysosomes are always damaged before cell fusion occurs regardless of the nature of the agent inducing fusion, and Allison and Mallucci (1965) have reported changes in the lysosomes of cells infected with NDV. They compared two strains of this virus. A highly cytopathic strain caused irreversible destruc-tion of lysosomes, release of enzymes and consequent cell damage, while a less cytopathic strain induced only transient lysosomal damage

Kohn and Fuchs (1969) showed that the cell-fusing abilities of NDV were not related to virulence, findings at first sight at variance with ours. Their methods involved the use of high multiplicities of virus, most of the cell surface being affected by virus during adsorption so that cell fusion was seen within 3 hr, before the formation of new virus.

Acknowledgements

We thank W H Allen for supplying virus strains and Professor A P Waterson for help The work was aided by grants from the Wellcome Trust and the Agricultural Research Council

References Asphn F D 1952 Immunization against Newcastle disease with a virus of low virulence (strain F) and observa-tions on subclinical infection in partially resistant fowls Vet Rec 64 245 Dobson N 1939 Newcastle Disease Proc 7th World's Poultry Cong Cleveland 250 Granoff A 1 959 Studies on mixed infection with Newcastle disease virus Virology 9 248 Kohn A and Fuchs P 1969 Cell fusion by various strains of Newcastle disease virus and their virulence J Virol 3 539

Reeve P and Waterson A P 1970 The growth cycle of avirulent strains of Newcastle disease virus Microbtos 5 5-9 Roizman B 1962 Polykaroycytosis Cold Sprang Harb Symp Quant Saul 27 327 Poste G H 1969 A study of virus-induced polykaryocytosis Ph D thesis University of Bristol Schloer G M and Hanson R P 1968 Relationship of plaque size and virulence for chickens of 14 represent-ative Newcastle disease virus strains J Vuol 2 40

Accepted 10 December 1969

57 NDV, cell fusion and haemadsorption

THE FACULTY PRESS 88 Regent Street Cambridge England

J. Hyg., Crank (1970), 68, 61 61 Printed in Great Britain

Growth in chick chorioallantoic membranes of strains of Newcastle disease virus of differing virulence

BY P. REEVE, MARGARET ROSENBLUM AND

D. J. ALEXANDER Department of Virology, Royal Postgraduate Medical School,

London, W.12

(Received 31 July 1969)

SUMMARY The growth of eight strains of Newcastle disease virus in chick embryo chorio-

allantoic membranes was studied by comparing, at different times after infection, the amounts of haemagglutinin released into the allantoic fluid (extracellular haemagglutinin) with that associated with the membrane (cell-associated haemag-glutinin). The virulence of the strains examined differed in that some killed chick embryos more rapidly than others. All strains released similar amounts of extra-cellular haemagglutinin and maximum titres were achieved about 12 hr. after infection. With virulent strains cell-associated haemagglutinin titres increased exponentially until the death of the host and maximum titres were much higher than those of extracellular haemagglutinin. With avirulent strains cell-associated haemagglutinin titres increased exponentially for only a limited time and titres were always lower than the titres of extracellular haemagglutinin.

Similar results were obtained when the titres of neuraminidase and viral ribo-nucleoprotein were measured during the growth of two virulent and two avirulent strains. Virulence appears to be associated with the continued intracellular accumulation of viral antigens.

INTRODUCTION Newcastle disease virus (NDV) has been suggested as a suitable virus for the

study of virulence (Waterson, Pennington & Allan, 1967). Many strains are available and their virulence, defined here as the ability to cause disease or death in a host, ranges from highly virulent strains, which are extremely pathogenic for chickens, kill chick embryos rapidly and destroy infected chick embryo fibroblast cells in culture, to strains isolated from apparently normal chickens which are not cytopathic and kill chick embryos infrequently or not at all. Virulence can be measured accurately and reproducibly in chick embryos and young chickens, and all strains grow readily in the chick embryo chorioallantoic membrane and release high titres of infectious virus into the allantoic fluid. They can, therefore, be cul-tured in quantity. Virus titres can be accurately and easily measured in terms of infectivity, haemagglutinin and neuraminidase activity. Besides this, NDV is not only an important animal pathogen in its own right, but is biologically akin to some important pathogens of man and other vertebrates, including mumps, the

62 P. REEVE, MARGARET ROSENBLUM AND D. J. ALEXANDER

para-influenzas, measles and distemper viruses : studies with NDV are thus relevant in a wide context.

Studies on the isolated virion show a surprising uniformity among strains : all those so far examined under the electron microscope look alike (Waterson & Cruickshank, 1963) and no major serological differences have been seen using serum neutralization or haemagglutination-inhibition (HAI) tests (W. H. Allan, unpublished observations) or in gel diffusion studies (Pennington, 1967).

Strains which differ in virulence for the chick embryo also differ, in parallel with this, in cytopathogenicity. Schloer & Hanson (1968) have shown, for example, that the most virulent strains for chick embryos produce the largest plaques on chick embryo fibroblast monolayers. This suggests that it is in the intracellular stages of virus development that strain differences might be found.

T. H. Pennington (unpublished observations) has reported differences in the quantity of haemagglutinin found in the chorioallantoic membranes of chick embryos infected with different strains of NDV, although with all strains sub-stantially similar quantities of haemagglutinin (HA) were released into the allantoic fluid. However, these findings were based on samples taken, for all strains, 40 hr. after inoculation of the chick embryos: thus no information was available on the rates at which the haemagglutinin was produced. We have, there-fore, measured the production of haemagglutinin in chorioallantoic membranes of chick embryos throughout one cycle of growth of several strains of NDV. We have compared these results with the titres of haemagglutinin released into the allantoic fluid and we have also measured the rates at which neuraminidase (Mucopoly-saccharide N-acetylneuraminylhydrolase) and viral ribonucleoprotein are pro-duced.

METHODS Viruses

The following strains of NDV were used: Herts 33 (Herts), received from W. H. Allan, Ministry of Agriculture, Fisheries

and Food, Central Veterinary Laboratory, Weybridge, Surrey. Isolated at Wey-bridge in 1933 by Dobson (1939).

Italien, received from Professor R. Rott, Institute of Virology, Giessen, Ger-many. Isolated in Italy at some date before 1949 (Schafer, Schramm & Traub, 1949).

Texas GB, (Texas), received from W. H. Allan; isolated in 1948 near Austin, Texas, U.S.A.

Field Pheasant, isolated at Weybridge in 1962 from pheasants; clinically more virulent than Herts 33 (W. H. Allan, personal communication).

Beaudette C, received from Professor R. Rott. This is a heat stable variant isolated from the Beaudette strain by Granoff (1959).

Strain F, received from W. H. Allan, isolated in England by Asplin in 1949 (Asplin, 1952).

Queensland V 4 (Queensland), received from W. H. Allan, isolated in 1966 by Rylie, in Brisbane, Australia (W. H. Allan, personal communication).

Growth of NDV in chick chorioallantoic membranes 63

Ulster, received from J. B. McFerran, Ministry of Agriculture for Ireland, Belfast. Isolated in 1966 in Northern Ireland from the faeces passed by healthy chickens.

The viruses were grown in the allantois of 10-day-old chick embryos incubated at 37° C., and virus-infected allantoic fluids were stored at — 70° C.

Growth cycles in chick embryo chorioallantoic membranes For each experiment about 50 10-day-old chick embryos were inoculated with

0.5 ml. of infected allantoic fluid containing about 109 Egg m50, and incubated at 37° C. At intervals thereafter three chick embryos were harvested and the allantoic fluid pooled. The chorioallantoic membranes were pooled after three washes with ice cold saline (0.15m). All samples were stored at — 70° C. until assayed.

Allantoic membranes were thawed and blended in homogenizers with motor-driven Teflon pestles to give 50 % (w/v) suspensions in phosphate buffered saline, pH 7.2 (PBS).

Haemagglutination titrations Plastic plates were used with 0.2 ml. volumes and 1 % (v/v) chicken erythro-

cytes; titres are expressed as the reciprocal of the last dilution giving complete agglutination.

Neuraminidase assays Sample volumes of 0.1 ml. were incubated at 37° C. for 10 min. with 0.1 ml.

fetuin and 0.3 ml. 0.2m-KH2PO4-Na2HPO4 buffer (pH 6.0). Fetuin was prepared from foetal calf serum by the method of Graham (1961). The N-acetyl neuraminic acid (NANA) released was assayed by the method of Aminoff (1961). Results are expressed as pg. NANA liberated per ml. of sample.

Incorporation of 3H-uridine The allantoic fluid of each egg at the start of the growth cycle was inoculated

with 100 pc. 3H-uridine 5-T (purchased from the Radiochemical Centre, Amer-sham).

The estimation of 311-uridine incorporated into ribonuclease-insensitive acid-insoluble material was based on the method of Plagemann (1968). For each sample,

ml. was diluted with 0.5 ml. 0.01 M sodium deoxycholate, 0.1 ml. ribonuclease (100 pg. per ml. in PBS) and incubated at 37° C. for 30 min.; 2.0 ml. 1 N perchloric acid was added and, after centrifugation at 1000 g for 10 min., the precipitate was washed three times with ice cold 5 % (w/v) trichloracetic acid. The precipitate was dissolved by adding 0.5 ml. 1 N sodium hydroxide and incubating at 60° C. for 30 min.: 0.2 ml. of this solution was added to 10 ml. of scintillation fluid con-taining 8.0 g. butyl PBD (Ciba Ltd), 80.0 g. naphthalene, 400 ml. oxitol (ICI Ltd) and 600 ml. toluene. Radioactivity was measured using an ABAC SL 40 Liquid Scintillation Spectrometer (Inter-technique Ltd) with an efficiency of about 35 %.

64 P. REEVE, MARGARET ROSENBLITM AND D. J. ALEXANDER

RESULTS

The growth of seven strains of NDV in chick embryo chorioallantoic membranes was examined by measuring the amount of haemagglutinin released into the allantoic fluid (extracellular haemagglutinin) and that still associated with the allantoic membrane (cell-associated haemagglutinin). The strains examined differed in virulence in that some killed chick embryos more quickly than others ; Table 1 shows the mean death time (MDT) of chick embryos inoculated with one minimum lethal dose. Herts was the most virulent strain examined, with an MDT of 49 hr. Neither Queensland nor Ulster consistently killed chick embryos, many of which survived even high inocula of these strains. With all strains an increase in haemagglutinin was first detected in the allantoic fluid about 4 hr. after inoculation, and thereafter titres increased exponentially until 12-14 hr. after inoculation (Fig. 1).

Table 1. Relative virulence of Newcastle disease virus strains (from Waterson, Pennington & Allan, 1967)

Mean death time Intracerebral Strain (hr.) index

Herts 49 1.88 Itahen 50 1.86 Texas 50 F80 Beaudette C 62 1.48 F 168 0.25 Queensland oo 0.25 Ulster co 0.00

Mean death time is the average time in which eggs inoculated with one minimum lethal dose of virus are killed. The intracerebral index is estimated from the time taken for chicks to die after intracerebral inoculation. The results are based on a scoring system in which the maximum index is 2 (100 % mortality in one day) and the minimum is 0 (no recorded symptoms after 8 days).

All strains produced approximately the same levels of extracellular haemagglutinin but marked differences were apparent when haemagglutinin associated with the allantoic membranes was assayed. With strains Herts, Italien and Beaudette C the production of cell-associated haemagglutinin continued exponentially until at least 20 hr. after inoculation and only diminished when at least 50 % of the chick embryos were already dead (Table 2). Cell-associated haemagglutinin titres of these virulent strains greatly exceeded those measured in the allantoic fluid at the end of the growth cycle.

Strains Queensland, Ulster and F, on the other hand, produced only low levels of cell-associated haemagglutinin and the rate of production appeared to diminish after approximately 12 hr. The titres of cell-associated haemagglutinin in these avirulent strains never exceeded those measured in the allantoic fluid.

All chick embryos inoculated with the strains Herts, Italien and Beaudette C had died after approximately 30 hr.; of the less virulent strains only F con-sistently killed the majority of chick embryos (90 % dead within 96 hr.) whereas


50 % of chick embryos inoculated with Queensland and Ulster survived for seven days after inoculation (Table 2).

Four strains (Herts, Texas, Queensland and Ulster) were examined in greater detail to see if other structural antigens making up the NDV virions, viz. neur-aminidase and ribonucleoprotein, also accumulated in the allantoic membranes infected with virulent virus but not in those infected with avirulent strains. The results obtained supported this possibility: 3H-uridine incorporated into ribo-nuclease-resistant, acid-insoluble material and neuraminidase titres both increased exponentially in allantoic membranes infected with the virulent strains Herts and

— Herts.

— •

• —

•

•

F

• to,. Le

• • ••

oo o

00 — Field Pheasant

• o

_ • •

_

Ulster

o o o o

— Italien

• —

Queensland •

• • ••

o _

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.

LA; V

• 111111111111111

_ Beaudette C •

• — .

• • • o

—

0

• •

iiiiiiiiiiilill

2 6 10 14 18 22 26 30

2 6 10 14 18 22 26 30 Hours after infection

Fig. 1. The comparison of cell-associated (0-0) and extracellular (•—•) haemagglutination titres during the growth of seven different strains of NDV in eggs.

5 tr VG 68

Reci

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al H

A t

itre

10,000

1,000

100

10

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1,000

100

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1,000

100

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10,000

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10


Texas until the embryos died (Fig. 2). We have assumed that the 311-labelled product is either viral RNA or ribonucleoprotein: both double-stranded RNA (Plagemann, 1968) and ribonucleoprotein (Kingsbury & Darlington, 1968) resist digestion with ribonuclease whereas single-stranded (cellular) RNA would be destroyed. In allantoic membranes infected with the avirulent strain Queensland, titres of neuraminidase and ribonuclease-resistant, acid-insoluble 3H-labelled material increased exponentially only for the first 14-16 hr. after infection and then levelled off; with strain Ulster the exponential phase extended rather longer but levelled off within 24 hr. after infection (Fig. 3).

Table 2. Percentage death of chick embryos in eggs inoculated with different strains of Newcastle disease virus

Tune after

inoculation (hr.)

Strains of NDV

Herts Italien Beau- dette C F

Queens- land Ulster

22 0 0 0 0 0 0 24 7.5 — 0 0 0 0 26 15 59 9 0 0 0 28 34 78 56 0 0 0 29 — 100 — 0 0 0 30 44 100 0 0 0 32 100 0 0 0 48 0 0 0 69 57 7 20 74 70 7 20 89 97 20 27 96 100 23 30

114 36 43 138 40 53 142 57 53

DISCUSSION

The results obtained show that some strains of Newcastle disease virus had higher titres of viral components associated with the chorioallantoic membrane than with the allantoic fluid, suggesting two discrete groups of strains; (1) Virulent strains which kill chick embryos rapidly and accumulate all the major viral components in the chorioallantoic membrane to titres higher than in the allantoic fluid, and (2) avirulent strains which kill embryos slowly or not at all and produce similar extracellular antigen titres to virulent strains but in which the intracellular accumulation of virus antigen is limited.

Our results show that virulence is associated with the continued exponential production of cell-associated viral antigens without concomitant release of mature virions, a process terminated only by the death of the chick embryo.

Newcastle disease virus, like other myxoviruses, is assembled at the cell surface and the envelope may consist of or contain cell membrane (Waterson, 1968). The modification of the cell membrane presumably has a drastic effect on the cell

(a) (a) •

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. . .

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Texas Herts

2 6 10 14 18 22 26 2 6 10 14 18 22 26 Hours after infection

Fig. 2. Comparison of the extracellular (.-.) and cell-associated (0-0) major antigens of two virulent strains of NDV during growth in eggs. (a) Haemagglutinm. (b) Material incorporating 3H-uridino, in an acid-insoluble RNase-resistant fraction. (c) Nouraminidase, as ug. NANA released.

permeability and metabolism. It could therefore be postulated that the strains of virus producing the most intracellular haemagglutinin would be the most virulent. The loss of ability to carry out cellular repair would enhance this effect and Wilson (1968) has shown that infection with the strain Texas GB, which is highly virulent, inhibits host cell protein synthesis in chick embryo fibroblasts.

Compans et al. (1966) in a morphological study showed that BHK21-F cells infected with SV5 virus accumulated viral ribonucleoprotein and disintegrated after extensive fusion although little mature virus was released. In contrast, primary rhesus monkey kidney (MK) cells infected with SV5 did not accumulate RNP, released infective virus, but were not destroyed. Virulence in their system

5-z


Queensland

Ulster

1000

1000 E

O

100

10 100

0

10

5

(a)

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.

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Fig. 3. Comparison of the extracellular (0-0) and cell-associated (Q—Q) major antigens of two avirulent strains of NDV during growth in eggs. (a) Haemagglutinin. (b) Material incorporating 31-1-uridine in an acid-insoluble RNase-resistant fraction. (c) Neuramimdase, pg. NANA released.

was determined by host cell differences but was also mediated by changes in the cellular surface and accumulation of viral antigen. In our system, unlike that stud-ied by Compans et al., virulence is determined by differences in the virus strain, the same host system being used throughout. Although avirulent strains did not appear to accumulate viral antigens in the chorioallantoic membrane we do not

Growth of N DV in chick chorioallantoic membranes 69

know if all the cells were infected. Fewer cells may have been infected by avirulent strains than by virulent strains, but since extracellular virus titres are similar with all strains this would require cells infected with avirulent viruses to release relatively more virus into the allantoic fluid than cells infected with virulent virus.

If the same number of cells is infected the difference between strains may be that the intracellular production of viral antigen is limited in cells infected with avirulent strains either by some host cell response or some limiting factor in the virus antigen production mechanism.

Margaret Rosenblum was a visiting student assistant supported by the Polio Research Fund (A.C.C.). D. J. Alexander is supported by the Agricultural Research Council. This research was aided by a grant from the Wellcome Trust. We are indebted to the British Egg Marketing Board for a grant for the eggs used. We acknowledge the technical assistance of Miss Joy Pacey and John Hall and we are most grateful to Professor A. P. Waterson for his help and encouragement.

REFERENCES

AMINOFF, D. (1961). Methods for quantitative estimation of N-acetylneurammic acid and their application to hydrolysates of sialomucoids. Biochemical Journal 81, 384.

ASvLIN, F. D. (1952). Immumsation against Newcastle disease with a virus of low virulence (strain F) and observations on sub-clinical infection in partially resistant fowls. Veterinary Record 64, 245.

COMPANS, R. W., HOLMES, KATHRYN V., DALES, S. & CFIOPPIN, P. W. (1966). An electron microscopic study of moderate and virulent virus-cell interactions of the paramfluenza virus SV5. Virology 30, 411.

DOBSON, N. (1939). Newcastle Disease. Proceedings of the 7th TVorld's Poultry Congress, Cleveland, p. 250.

GRAHAM, E. R. B. (1961). Some aspects of the structure of the carbohydrate moiety of fetuin. Australian Journal of Science 24, 140.

GRANOFF, A. (1959). Studies on mixed infection with Newcastle disease virus. Virology 9, 636. KINGSBURY, D. W. & DARLINGTON, R. W. (1968). Isolation and properties of Newcastle

disease virus nucleocapsid. Journal of Virology 2, 248. PENNINGTON, T. H. (1967). Studies with Newcastle disease virus. Ph.D. Thesis, University of

London. PLAGEMANN, P. W. (1968). Mengovirus replication in Novikoff rat hepatoma and mouse L

cells: Effects on synthesis of host-cell macromolecules and virus specific synthesis of ribonucleic acid. Journal of Virology 2, 461.

ScHAFER, W., SCHRAMM, G. & TRAUB, E. (1949). Untersuchungen uber das Virus der atyp-ischen Geflugelpest. Zeitschrift fur Naturforschung 46, 157.

SCHLOER, G. M. & HANsoN, R. P. (1968). Relationship of plaque size and virulence for chickens of 14 representative Newcastle disease virus strains. Journal of Virology 2, 40.

WATERSON, A. P. (1968). Introduction to Animal Virology. 2nd ed., Cambridge University Press.

WATERSON, A. P. & CRUICKSHANK, J. G. (1963). The effect of ether on Newcastle disease virus: a morphological study of eight strains. Zeitschrift fur Naturforschung 18B, 114.

WATERSON, A. P., PENNINGTON, T. H. & ALLAN, W. H. (1967). Virulence in Newcastle disease virus. A preliminary study. British Medical Bulletin 23 (2), 138.

WILSON, D. E. (1968). Inhibition of host-cell protein and RNA synthesis by Newcastle disease virus. Journal of Virology 2, 1.

J. gen. Viral. 0970, II, 25-34 25 Printed in Great Britain

Studies on the Cytopathic Effects of Newcastle Disease Virus: Metabolic Requirements

By P. REEVE, D. J. ALEXANDER, GILLIAN POPE AND G. POSTE

Department of Virology, Royal Postgraduate Medical School, Du Cane Road, London, W12, England

(Accepted 17 December 1970)

SUMMARY

Nine strains of Newcastle disease virus were examined for their ability to inhibit cellular protein synthesis and to cause cell fusion. Inhibition of cellular protein synthesis was confined to infection with virulent strains (HERTS, WARWICK, TEXAS, `H', FIELD PHEASANT) and the mesogenic strain BEAUDETTE C. No inhibition of synthesis was recorded with avirulent strains (ULSTER, F, QUEENSLAND). Inhibition of cellular protein synthesis required virus protein synthesis within 3 hr of infection and could be inhibited by parafluorophenylalanine and Congo red. Cell fusion and haemadsorption by the various Newcastle disease virus strains also required virus-induced protein synthesis and were inhibited by cycloheximide, parafluorophenyl-alanine and Congo red. However, neither virus-induced inhibition of cellular protein synthesis nor cell fusion required new virus RNA synthesis, since azauridine did not affect these processes. The importance of virus-induced proteins in the inhibition of protein sythesis and cell fusion is discussed.

INTRODUCTION

Previous studies (Reeve & Alexander, 1970; Reeve & Poste, 1971) have shown that the formation of polykaryocytes by cell fusion is the initial cytopathic effect induced by New-castle disease virus (NDV) grown in chick embryo cells. These studies on NDV also showed that there was a direct relation between virulence and the capacity of strains to cause cytopathic effect. In parallel with cytopathic effect many viruses also produce profound alterations in cellular metabolism, particularly by the inhibition of the synthesis of host cell macromolecules (for review, see Roizman & Spear, 1969). However, only limited informa-tion is available on the effect of NDV on host cell macromolecular synthesis. Wilson (1968) found that the virulent strain TEXAS inhibited host cell RNA and protein synthesis, while the mesogenic strain BEAUDETTE c inhibited only protein synthesis. The inhibition of host cell protein synthesis induced by NDV occurs relatively late in the virus growth cycle, in contrast to the 'early' inhibition induced by the picorna-, adeno- and poxviruses (Martin & Kerr, 1968). The significance of NDV-induced inhibition of cellular macromolecular synthesis is not clear, nor is it known whether this inhibition is related to the virulence of the infecting strain. Several strains of NDV of distinct virulence have therefore been examined for their ability to inhibit cellular protein synthesis. Experiments were also done with selective metabolic inhibitors to test whether the ability of NDV to inhibit protein synthesis and to cause cytopathic effect were linked through a common mechanism.

26 P. REEVE AND OTHERS

METHODS

Virus strains and cell culture. The cell culture techniques and most of the virus strains have been described (Reeve & Waterson, 1970; Reeve, Rosenblum & Alexander, 1970). Strains H' and WARWICK were obtained from W. H. Allan Central Veterinary Laboratories, Wey-

bridge, Surrey. All experiments were done on coverslip cell cultures. Chick embryo cell monolayers were inoculated with 0.5 ml. virus-infected allantoic fluid diluted in maintenance medium (Eagle's BH K supplemented with 2 % foetal calf serum, Burroughs Wellcome Ltd., Beckenham, Kent) to provide a multiplicity of infection of 5o infective particles/cell. After adsorption at 37° for 1 hr cultures were washed three times with pre-warmed phosphate-buffered saline (PBS) to remove any remaining virus and fresh maintenance medium was added with metabolic inhibitors or radioisotopes as appropriate. Cultures were then in-cubated at 37° in the stationary position.

Metabolic inhibitors. Congo red, azauridme, cycloheximide and p-fluorophenylalanine (FPA) were obtained from Koch—Light Laboratories, Colnbrook, England and used in cell culture media as freshly prepared solutions at the appropriate concentrations.

Radioisotopes. [3H]L-leucine 4,5-T (19.7 c/mM) and [3H]uridine 5-T (26.5 c/mm) were obtained from the Radiochemical Centre, Amersham, Buckinghamshire.

Incorporation of radioisotopes. The rates of protein and RNA synthesis were estimated by measuring the incorporation of radioactive precursors into trichloroacetic acid-insoluble material. Maintenance medium containing 1-0 pc/ml. isotope was used to pulse coverslip cell cultures for 3o or 6o min. The coverslips were then washed once with ice-cold PBS, four times with ice-cold 5 % (w/v) trichloracetic acid and once in acetone. The coverslips were air-dried, and the acid-insoluble radioactivity estimated in an Intertechnique ABAC SL 4o liquid scintillation spectrometer using toluene containing 8-0 g./1. butyl PBD as scintillation fluid.

Estimation of cell fusion as % polykaryocytosis. The extent of cell fusion produced by virus strains was estimated 15 hr after infection from counts on stained coverslip cell cultures (Reeve & Poste, 1971).

Estimation of haemadsorption. Coverslip cell cultures were washed three times with ice-cold PBS 8 hr after infection and then received 0.5 ml. of a o•5 % (v/v) suspension of chicken erythrocytes. After 15 to 20 min. at 4°, unadsorbed erythrocytes were removed by three further washes with ice-cold PBS. The cultures were then fixed in methanol, stained with May—Griinwald Giemsa (Jacobson & Webb, 1952), and the number of erythrocytes adsorbed to the cells was counted. The amount of haemadsorption is expressed in terms of the number of erythrocytes in cultures treated with metabolic inhibitors expressed as a percentage of the number in inhibitor free cultures. Uninfected chick embryo cell cultures did not adsorb erythrocytes.

Estimation of cellular permeability. The permeability of infected cells was estimated by assaying the amount of lactate dehydrogenase released from the cells into the culture medium. For this assay, cell cultures were grown in 15 cm. glass Petri dishes and the lactate dehydrogenase released was estimated spectrophotometrically (Kornberg, 1955) and ex-pressed as a percentage of the total lactate dehydrogenase present.

4 8 12 16 20 24 4 8 12 16 20 24 Hr after infection

NDV cytopathic effect 27

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Fig t Incorporation of [31-I]leucine into trichloracetic acid-insoluble fractions of chick embryo cells infected with different strains of NDV (•—•). The results are expressed as of the incorporation into uninfected control cells. The release of lactate dehydrogenase from chick embryo cells infected with NDV strains (—E1), is expressed as a percentage of the total cellular lactate dehydrogenase.

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RESULTS

Relation between virulence of the infecting strain and inhibition of cellular protein synthesis

The virulent Newcastle disease virus strains HERTS, WARWICK, `LI', TEXAS and FIELD PHEASANT and the mesogenic strain BEAUDETTE c all caused marked inhibition of protein synthesis, as judged by the ability of the infected cells to incorporate [3H]-leucine in tri-chloroacetic acid-insoluble material after a 3o min. pulse when compared with the uninfected control cells (Fig. I). All of these strains inhibited cellular protein synthesis by 5o% at 6 to 8 hr after infection, at which time new infective virus appeared in the medium and 5o % of the final yield of mature virus had been synthesized (Reeve & Waterson, 1970). The avirulent vaccine strain F inhibited cellular protein synthesis by about 40 %, but this was not detected until 18 hr after infection. In contrast, the avirulent strains QUEENSLAND and ULSTER did not inhibit cellular protein synthesis.

100 200 300 400 100 200 300 400

Fluorophenylalanme Gig /ml )

Fluorophenylalanine (ig /ml )

Fig 2 (a). Prevention by p-fluorophenylalanine of the inhibition of cellular protein synthesis in chick embryo cells by strains HERTS (0-110) and 're (II—E).Fluorophenylalanine was added' hr after infection to control and infected cultures 7 hr later cultures were pulsed for 1 hr with PHI. leucine and incorporation into an acid insoluble fraction was measured The incorporation of isotope by infected cultures was compared with that of corresponding uninfected control cultures in the presence of fluorophenylalanine and results are expressed as % inhibition of incorporation (b) The effect of different concentrations of fluorophenylalanine on haemadsorption (•—A) and cell fusion (•—•), by chick embryo cells infected with strain HERTS. i hr after infection fluoro-phenylalanine was added, haemadsorption and cell fusion were measured 8 and 15 hr after infection, respectively. The results arc expressed as °/;) of the amount of haemadsorption or cell fusion in infected cultures without fluorophenylalanine

Relation between inhibition of cellular protein synthesis, cytopathic effect and cell damage

All the strains which inhibited cellular protein synthesis induced extensive cell fusion and damage. Cytopathic effect, in the form of cell fusion, was first seen 6 to 8 hr after infection at the time when 5o % inhibition of cellular protein synthesis was recorded. Inhibition of cellular protein synthesis might then be merely a non-specific effect secondary to cell damage and to the development of cytopathic effect. We therefore compared the rate at which inhibition of protein synthesis occurred with the rate at which the enzyme lactate dehydro-genase was lost from the cell through the damaged plasma membrane. With two virulent


strains, HERTS and TEXAS and the avirulent QUEENSLAND and F strains, a slow loss of lactate dehydrogenase was recorded during the first i6 hr after infection. After this a rapid increase in lactate dehydrogenase release, indicative of extensive cellular damage, was seen only in cells infected with the virulent strains (Fig. 1). These results suggest that gross cellular damage produced by virus replication occurred at 16 to 24 hr after infection and too late to account for the marked inhibition of protein synthesis found 6 to 8 hr after infection.

Effect of inhibitors of protein synthesis on virus-induced inhibition of protein synthesis, cell fusion and haemadsorption

From these experiments we concluded that inhibition of cellular protein synthesis was related directly to the virulence of the infecting strain of NDV. Other properties of the virus which are related directly to its virulence include the ability to produce cell fusion (Reeve & Poste, 197o) and haemadsorption (Reeve & Alexander, 1970). We therefore examined these three properties to determine whether there was a common requirement for protein synthesis.

Cell cultures were infected with NDV strains in the presence of the following metabolic inhibitors and the inhibition of cellular protein synthesis, extent of cell fusion and the degree of haemadsorption measured.

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80

60

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20

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80

Congo Red (pg /ml )

20 40 60 80

Congo Red (pg Iml)

Fig. 3 (a). Prevention by Congo red of the inhibition of cellular protein synthesis in chick embryo cells by strain HERTS. Congo red was added to control and infected cultures 1 hr after infection. Cultures were pulsed 7 hr later fort hr with [31-1]leucine and incorporation into acid-insoluble material measured The incorporation of isotope by infected cultures was compared with that of corresponding uninfected control cultures in the presence of Congo red and results are expressed as

inhibition of incorporation observed. (b). Effect of different concentrations of Congo red on haemadsorption (♦—A) and cell fusion (■—■) by chick embryo cells infected with strain BER-rs. Congo red was added i hr after infection, and haemadsorption and cell fusion measured 8 and 15 hr after infection, respectively The results are expressed as of the amount of haemadsorption or cell fusion in infected cultures without Congo red.

p-Fluorophenylalanine, which inhibits protein synthesis by replacing phenylalanine to form non-functional proteins, prevented the inhibition of cellular protein synthesis by strains HERTS and `1-1' (Fig. 2a) and also prevented cell fusion and haemadsorption by strain HERTS (Fig. 2h).

Congo red. Becht & Drzeniek (1968) described the inhibition of NDV replication by this agent. Doses of io pg./ml. or greater inhibited the synthesis of virus proteins, haemagglutinin

3 vig II


and neuraminidase, but ribonucleoprotein synthesis was unaffected. In our system, Congo red prevented cell fusion, haemadsorption and inhibition of cellular protein synthesis by strain HERTS (Fig. 3a, b). However, these results were complicated by the toxicity of doses of 8o—too //g./ml. for chick embryo cell monolayers, particularly when the dye was in contact with the cells for 15 hr. In addition, in uninfected control cells treated with this compound, an inhibition of protein synthesis indicated that Congo red may act as a general inhibitor of protein synthesis, rather than in the specific way described by Becht & Drzeniek (1968).

Cycloheximide. At concentrations of 5 fig./ml. or greater, cycloheximide inhibited protein synthesis in uninfected cells within 3 hr (Fig. 4). In cells infected with strain HERTS, similar doses of cycloheximide inhibited both cell fusion and haemadsorption (Fig. 4).

100

80

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O ON-2

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0 20 40 60

80

Cycloheximide (Fig /ml )

Fig. 4 Effect of different concentrations of cycloheximide on protein synthesis in uninfected chick embryo cells (•—•) and on haemadsorption (♦—A) and cell fusion (■—■) in chick embryo cells infected with strain HERTS Protein synthesis was measured 3 hr after the addition of cyclo-heximide by the incorporation of PH]leneine into an acid-insoluble fraction Cycloheximide was added i hr after infection and haemadsorption and cell fusion were measured 8 and 15 hr after infection, respectively All results are expressed as °,„' of activity in controls without cycloheximide.

These results with inhibitors of protein synthesis showed that all the features of the cytopathic effect produced by virulent NDV strains; inhibition of cellular protein synthesis, formation of polykaryocytes by cell fusion and formation of haemadsorption centres required the synthesis of virus-induced proteins.

The effect of time of addition of inhibitors of protein synthesis on virus-induced inhibition of protein synthesis, cell fusion and haemadsorption

In order to determine the time in the growth cycle at which the protein or proteins concerned with the expression of cytopathic effect were produced, cells were infected with strain HERTS, and at intervals after infection either 500 fig./ml. fluorophenylalanine or 50 pg./mi. Congo red was added. At To hr after infection the cultures were washed and inhibitor-free medium with [3H]leucine was added. After incubation for i hr at 37°, the

1 2 3 4 5 6 7 8 9 10 Hr after infection

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16 Hr after infection

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incorporation of the isotope was measured and the extent to which cellular protein synthesis had been inhibited by virus infection was estimated.

The addition of either inhibitor 2 hr after infection prevented the inhibition of cellular protein synthesis by the virus, but after that time the inhibition increased (Fig. 5). Thus, it appears that protein synthesis is required within 3 hr of infection for virus-induced inhibition of cellular protein synthesis to occur. A concentration of 500 €g.Jml. fluorophenylalanine prevented cell fusion and haemadsorption by strain HFRTS when added within 2 hr of infection, but not when added over 4 hr after infection (Fig. 6). Experiments with strains ' and WARWICK gave similar results.

We conclude that protein synthesis must occur very early in the growth cycle for cyto-pathic effect to occur, and that the protein or proteins implicated in this cytopathic effect must be synthesized within 3 hr of infection.

Fig 5 Fig. 6

Fig. 5. Effect of adding fluorophenylalanine (5oo pg ml , 0-0) or Congo red (5o pg , •—•) at different times after infection on the degree of inhibition of cellular protein synthesis by strain HERTS At intervals after infection fluorophenylalanine or Congo red was added to infected cultures and uninfected control cultures All cultures were pulsed with [ 31-illeticine for i hr at to hr after infection and incorporation into an acid-insoluble fraction was measured The results are expressed as % inhibition of protein synthesis calculated from the amount of incorporation of [3H]leucine by uninfected cultures The points indicate the times after infection at which inhibitor was added Fig 6. Effect of adding fluorophenylalanine (500 pg ml) at different times after infection on haemadsorption ( A.—•) and cell fusion (•—•) in chick embryo cells infected with strain HERTS. The points indicate the times after infection at which fluorophenylalanine was added. Haemad-sorption and cell fusion were estimated 8 and 15 hr after infection, respectively The results are expressed as of those for infected cultures without fluorophenylalanine

Effect of inhibition of RNA synthesis on cytopathic effect Since the protein synthesis associated with the development of cytopathic effect occurred

early in the growth cycle, it suggested that the RNA of the infecting particle, rather than newly synthesized RNA, was used directly for coding this synthesis. To test this hypothesis, azauridine was used to block RNA synthesis. Wilson & Lo Gerfo (1964) showed that azauri-dine inhibited the synthesis of virus and cellular RNA to the same extent. In our system, azauridine at 90 pg./ml. inhibited cellular RNA synthesis by over 50 %, as shown by the

3-2


incorporation of [3H]uridine into uninfected cells. At woo itg./ml. or greater cellular RNA synthesis was inhibited by at least 90 % (Fig. 7). However, cell fusion and haemadsorption in cells infected with strain HERTS were unaffected by up to 3000 azauridine (Fig. 7).

Activit

y as

% o

f th

at in c

on

tro

ls w

ithou

t aza

uri

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e

1 2

3 Azauridine (mg /ml )

Fig 7. Effect of different concentrations of azauridine on RNA synthesis in uninfected chick embryo cells (•—s) and on haemadsorption (A—A) and cell fusion (■—■) in chick embryo cells infected with strain HERTS RNA synthesis was measured by the incorporation of [3H]uridine into an acid-insoluble fraction 3 hr after the addition of azauridme. Azaurichne was added 1 hr after infection and haemadsorption and cell fusion measured 8 and 15 hr after infection, respectively. Results are expressed as °,10 of activity in cultures without azauridine.

DISCUSSION

Our results show that the ability of Newcastle disease virus strains to inhibit cellular protein synthesis is related directly to the virulence of the infecting strain. Inhibition of cellular protein synthesis by NDV strains occurred fairly late in the growth cycle. A 5o % inhibition was recorded 6 to 8 hr after infection, by which time 5o % of the final yield of mature virus had already been synthesized (Reeve & Waterson, 1970). It is therefore unlikely that the inhibition produced by NDV is comparable with the rapid inhibition seen with the picornaviruses and vaccinia virus (Martin & Kerr, 1968; Bablanian, 1970). However, since extensive leakage of lactate dehydrogenase from infected cells was not seen until some hr after inhibition of protein synthesis, it is unlikely that inhibition results merely from gross cellular damage, but must be due to an early event induced by virus. The results obtained using inhibitors showed that some protein synthesis was required early in the virus growth cycle if cellular protein synthesis was to be inhibited subsequently. The other manifestations of virus infection, haemadsorption and cell fusion, were also dependent upon early protein synthesis.

A requirement for protein synthesis during virus-induced cell fusion has been demonstrated with other viruses. In vaccinia virus-infected L cells, puromycin inhibited the formation of polykaryocytes by cell fusion (Kaku & Kamahora, 1964). Falke (1965, 1967) found that

NDV cytopathic effect 33 cycloheximide and fluorophenylalanine, but not chloramphenicol, prevented the fusion of rabbit kidney cells by herpes simplex virus. More recently, Bratt & Gallaher (1969) showed that cycloheximide prevented cell fusion in chick embryo cells infected with the VICTORIA

strain of NDV. It is not clear whether the proteins involved in cell fusion by NDV are structural com-

ponents of the virus particle or how they act to induce fusion. The labilization of lysosomes is known to be important in producing the cell surface changes necessary for fusion (Allison, 1967; Poste, 1970; Poste & Allison, 1971), and the possibility must be considered that the virus-induced proteins involved in fusion act on the lysosome system. There are indications from the other virus—cell systems that virus-induced proteins are responsible for specific labilization of lysosomes (Amako & Dales, 1967; Thacore & Wolff, 1968; Guskey, Smith & Wolff, 1970). Furthermore, Allison & Mallucci (1965) showed that virulent strains of NDV produced extensive and permanent damage to lysosomes, while avirulent strains induced only transient and reversible damage.

Although our results do not permit a strict distinction between the cellular events involved in the inhibition of protein synthesis and those in cell fusion, it is unlikely that both are mediated by the same mechanism, even though the same proteins may be implicated.

Newcastle disease virus induces the synthesis of both RNA complementary to that of the virus particle, — RNA, and replicas of the virus genome, + RNA. In the first 7 hr of infection of chick embryo cells by BEAUDETTE c, up to 90 % of virus RNA is negative-stranded (Kings-bury, 1970). The functions of the different RNAs are not clear: Kingsbury (1966) and Bratt & Robinson (1967) suggested that the —RNA acts as messenger for some or all virus proteins. However, Schafer, Pister & Schneider (1967) showed that virus antigens could be synthesized in chick embryo cells infected with BEAUDETTE C in the presence of azauridine, and concluded that parental + RNA acted as messenger. Since both virus-induced cell fusion and haemadsorption were unaffected by azauridine, our results support the suggestion of Schafer et al. (1967) that parental RNA can act as messenger. Long & Burke (1970), using u.v. irradiation to inhibit RNA synthesis, also concluded that the parental strand of fowl plague RNA acted directly as messenger for the proteins involved in the inhibition of host cell protein synthesis. Recent studies have also shown that some virus-induced proteins were synthesized in Sendai virus infected cells using the RNA of the infecting particle as mess-enger (Mekler, Shlyankevitch & Shevliaghyn, 1970). It is important to confirm, therefore, whether the nucleic acid of the virus particle can act directly as a messenger for the proteins involved in cytopathogenicity, since our work implies that the synthesis of nucleic acid and the subsequent assembly and release of mature particles may be irrelevant to the expression of virus-induced cell damage.

This research was aided by grants from the Wellcome Trust, The Agricultural Research Council and Action for the Crippled Child (Polio Research Fund). D. A. was supported by the Agricultural Research Council, G. P. by the Cancer Research Campaign and P. R. by the Wellcome Trust. We are most grateful to Professor A. P. Waterson for his help and encouragement throughout this work and in the preparation of the paper.

34

P. REEVE AND OTHERS

REFERENCES

ALLISON, A c (1967). Lysosomes in virus infected cells Perspectives in Virology 5, 29 ALLISON, A c & MALLUCCI, L (1965) Histochemical Studies of lysosomes and lysosomal enzymes in virus-

infected cell cultures. Journal of Ever 'menial Medicine 12i, 463. AMAKO, K & DALES, S (1967) Cytopathology of mengovirus infection. I Relationship between cellular

disintegration and virulence. Virology 32, 201 BABLAN1AN, R (1970). Studies on the mechanism of vaccinia virus cytopathic effects. effect of inhibitors of

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(Received 24 July 1970)

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