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IN VIVO AND IN WITRO STUDIES OM BOVINE PAPILLOMA VIRUS

by

H e iko R o g ie r C h r i s t i a a n MEISCHKE ELU.S c * , M«VoSc., MRCVS,

A t h e s i s s u b m i t te d t o th e U n i v e r s i t y o f Glasgow i n p a r t i a l

f u l f i l m e n t o f th e re q u i re m e n ts le a d in g to th e deg ree o f D o c to r

o f P h i lo s o p h y *

D epartm en t o f V e t e r i n a r y P a th o lo g y

F a c u l t y o f V e t e r i n a r y M e d ic in e

U n i v e r s i t y o f Glasgow

Septemberp 1978*

O 1 tt

ProQuest Number: 10656464

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IS

r T ’ SGOWL NIVERSITYl ib r a r y

TABLE OF CONTENTS

Page

PRELIMINARIES

Table of contents 2List of tables 8List of figures 10List of abbreviations 12Acknowledgements 14-Declaration 16Photocopy restrictions 16Summary 17

Chapter 1 LITERATURE REVIEW AND GENERAL INTRODUCTION1*1 The Papovaviridae 20

1,1„1 General properties of the papovaviruses 201 * 1 ,2 Original isolation of the papovaviruses 20

1.2 Polyomaviruses in Vivo - Host Range andOncogenicity 24

1 *2 ,1 Polyomavirus 251 . 2 . 2 SV40 261 . 2 . 3 BKV, RFV, and MMV 2 ?

1 .2 .4 :jcv 2 9

1 . 2 . 5 O th e r v i r u s e s of th e polyoma group 501 .3 Papillomaviruses in Vivo - Most Range

511 ,3 .1 Human p a p i l lo m a v i r u s 511 . 3 . 2 V e r ru c a v u l g a r i s , p la n a , p l a n t a r i s 521 .3 .3 Condyloma a cu m in a ta 551 , 3 , 4 A l im e n ta r y and r e s p i r a t o r y t r a c t p a p i l l o m a t o s i s 541 . 3 . 5 E p id e rm o d y s p la s ia v e r r u c i f o r m i s 561 , 3 . 6 Shops p a p i l lo m a v i r u s 571 .3 .7 R a b b i t o r a l p a p i l l o m a v i r u s 571 .3 .8 C an ine p a p i l lo m a v i r u s 581 . 3 . 9 C an ine o r a l p a p i l l o m a v i r u s 581 .3 .1 0 E qu ine p a p i l lo m a v i r u s 581 ,3 .1 1 Goat p a p i l l o m a v i r u s 581.3 .1 2 Ham ster p a p i l l o m a v i r u s 581 .3 .1 3 Sheep p a p i l lo m a v i r u s 591 .3 .1 4 Deer f ib r o m a v i r u s 591 .3 .1 5 Equ ine s a r c o id 59

2,

1*4

1 .5

1 . 6

o3o16 B ov ine p a p i l lo m a v i r u s

.3 ,1 7 O th e r p a p i l lo m a v i r u s e s

«3,18 Summary - p a p o v a v i ru s e s i n v i v o

P a p o v a v iru s e s i n V i t r o

.4 ,1 Polyoma and SV40

. 4 . 2 BKV, RFV, MMV

«4,3 3CV

. 4 . 4 O th e r p o ly o m a v iru s e s i n v i t r o

,4o5 Human p a p i l lo m a v i r u s

o4o6 Shope p a p i l lo m a v i r u s

*4o7 B ov ine p a p i l lo m a v i r u s

*4oB O th e r p a p i l l o m a v i r u s e s i n v i t r o

Im m u n o lo g ic a l A sp e c ts o f P a p o v a v iru s I n f e c t i o n

1 .7

1.8

,5 „1 P o ly o m a v iru s e s

. 5 . 2 Human p a p i l lo m a v i r u s

«5 .3 Shope p a p i l lo m a v i r u s

«5 .4 B o v in e p a p i l lo m a v i r u s

«5 .5 O th e r p a p i l l o m a v i r u s e s

„5o6 Summary - im m u n o lo g ic a l a sp e c ts

Phys icD - C hem ica l C h a r a c t e r i s t i c s o f th e Papoya■

y j r i d a e

.6 .1 P a p o v a v iru s h a e m a g g lu t in a t io n

. 6 . 2 P a p o v a v i ru s p a r t i c l e d ia m e te r

, 6 . 3 T h e r m o s t a b i l i t y o f th e p a p o v a v iru s e s

Summary o f th e L i t e r a t u r e Review

G e n e ra l I n t r o d u c t i o n

Page

4-0404043434546464748 484849 49 52 545455 55

555657 59 59 64-

C h a p te r 2

2 .1

2 . 2

2 .2.1

2.2 . 2

2 .2 .3

2 .2 .4

2 . 2 . 5

2 .2 . 6

IN VIVO EFFECTS OF BOVINE PAPILLOMA VIRUS :

AN ABATTOIR SURVEY OF BOVINE PAPILLOMATOSIS AND

COMPARISON BY ELECTRON MICROSCOPY AND ELECTROPHORESIS

OF BPV ISOLATED FROM VARIOUS LESION TYPES

I n t r o d u c t i o n

M a t e r i a l s and Methods

C a t t l e S urvey

V i r u s e x t r a c t i o n

P a p i l lo m a c l a s s i f i c a t i o n

E le c t r o n m ic r o s c o p ic e x a m in a t io n

V i r u s measurement

P o ly a c r y la m i d e g e l e l e c t r o p h o r e s i s

66

66

6666

6767677171

o 3,

Page

2 * 2 ,7 P a p i l lo m a s c r a p in g y g

2 ,3 R e s u l t s

2 ,4 D is c u s s io n7287

2 ,5 Summary o n

C h a p te r 3 IIM VIVO EFFECTS OF BOVINE PAPILLOMA VIRUS :

EXPERIMENTAL TRANSMISSION TO CATTLE OF BPV

EXTRACTED FROM MORPHOLOGICALLY SEPARABLE TEAT

AND CUTANEOUS LESIONS AND THE EFFECTS OF

INOCULATION OF BPV - TRANSFORMED FOETAL BOVINE

CELLS. 923o1 I n t r o d u c t i o n g 2

3 . 2 M a t e r i a l s and Methods gg

3 .2 .1 L e s io n s g2

3 . 2 . 2 V i r u s e x t r a c t i o n 93

3 . 2 . 3 F o e ta l b o v in e c e l l s 9 3

3 . 2 . 4 A n im a l i n o c u l a t i o n gzj.

3 . 2 . 5 B io p s y sam ples 953 . 2 . 6 C h a l le n g e e x p e r im e n t g g

3 . 2 . 7 P o s t -m o r te m e x a m in a t io n g g

3 . 2 . 8 H is to p a th o lo g y g^^

3 . 3 _____ R e s u l t s g g

3 .3 .1 H is t o p a th o lo g y g g

3 . 3 . 2 C h a l le n g e 2.09

3 . 3 . 3 E le c t r o n m ic ro s c o p y 1123 . 3 . 4 P o s t -m o r te m e x a m in a t io n

3 .4 D is c u s s io n112114

3 ,5 Summary 1 1 8

C h a p te r 4 IN VIVO EFFECTS OF BOVINE PAPILLOMA VIRUS :

THE TUMOURIGENICITY IN ATHYMIC NUDE MICE OF

BOVINE PAPILLOMA VIRUS AND BPV-TRANSFORMED

FOETAL BOVINE SKIN CELLS 1194 .1 I n t r o d u c t i o n I - | o

4 .2 M a t e r i a l s and Methods 1n q

4 .2 .1 C e l l s 1194 . 2 . 2 E x a m in a t io n o f c e l l s 12Q

4 . 2 . 3 V i r u s -120.

4 . 2 . 4 Nude m ice 1 2 0

,4,

Page

4 * 2 ,5 H is to p a th o lo g y ^224o2o6 E le c t r o n m ic ro s c o p y 2,224 , 2 . 7 A t te m p ts a t i n v i v o and i n v i t r o p ro p a g a t io n 122

o f tum ours

4 ,3 R e s u l t s i

4 .4 D is c u s s io n 1294 ,5 Summary 131

C h a p te r 5 IN VIVO EFFECTS OF BOVINE PAPILLOMA VIRUS :

THE EFFECTS OF INTRACEREBRAL INOCULATION OF BOVINE

PAPILLOMA VIRUS AND BPV-TRANSFORMED CELLS IN

EXPERIMENTAL CALVES 1325o 1 I n t r o d u c t i o n 132

1325 .2 M a t e r i a l s and Methods

5 .2 .1 E x p e r im e n ta l c a lv e s 1325 . 2 . 2 V i r u s 1335 . 2 . 3 C e l l s 134

5 .3 R e s u l ts 1341375 .4 D is c u s s io n

5 .5 Summary 139

C h a p te r 6 THE EFFECT OF PASTEURISATION ON BOVINE

PAPILLOMA VIRUS IN VIVO AND IN VITRO 1406.1 I n t r o d u c t i o n 1406,2 M a t e r i a l s and Methods 140

6, 2.1 C l a s s i f i c a t i o n o f l e s i o n ty p e s 1406. 2.2 V i r u s e x t r a c t i o n 1416 . 2 . 3 E le c t r o n m ic r o s c o p ic e x a m in a t io n 1416 * 2 .4 P a s t e u r i s a t i o n 1416 .2 ,5 T e m p e ra tu re - t im e i n a c t i v a t i o n 1426. 2.6 I n v i v o t i t r a t i o n 1426 .2 .7 M i l k samples 143

6 ,3 R e s u l t s 1436 ,4 D is c u s s io n 1456 ,5 Summary 148

C h a p te r 7 IN VITRO TRANSFORMATION BY BOVINE PAPILLOMA VIRUS 1507,1 I n t r o d u c t i o n 150

, 5 ,

Page

7 . 2 _________ M a t e r i a l s and Methods ]_51

7 .2 .1 Primary cell cultures 1517.2.2 Skin calls 1517.2.3 Meningeal cells 1^27.2.4 Virus 1537 . 2 . 5 Infection of cells in suspension i g ' J

7.2.6 Infection of cells as monolayer 15^7 . 2 . 7 Examination of plates 1537 . 2 . 8 Calculation of Log TD50 1537 . 2 . 9 Characterisation of transforming cell cultures 1557 .2 .1 0 Adsorption time 1557 .2 .1 1 G e n e ra t io n i n t e r v a l 1 5 5

7.2.12 Estimation of cell longevity 1567,2o13 Agar suspension culture 1567o2o14 Haemagglutination 156

7 .3 Results 1567. 4 Discussion 1637 .5 Summary 167

C h ap te r 8 INHIB IT ION OF IN VITRO TRANSFORMATION BY BOVINE

PAPILLOMA VIRUS 1698.1 Introduction 1698.2 Materials and Methods 170

8,2o1 Virus, cells, sera and complement 1708.2.2 Varying complement and virus concentrations 1708 . 2 . 3 The effect of high titre sera on previously

transformed cell cultures 1728 . 2 . 4 Adsorption of high titre sera with various

BPV-related suspensions8 . 2 . 5 Investigation of effect of non-transforming

isolates8 .3 R e s u l t s ]_77

8.4 Discussion 1828.5_____ Summary 185

Chapter 9 ATTEMPTS TO GROW BOVINE PAPILLOMA VIRUS INVITRO

9,1 I n t r o d u c t i o n 186

« 6 ,

172

175

9.2

9 . 2.1

9 . 2.2

9 .2.3

9 . 2 . 4

9 .3

9.4

M a t e r i a l s and Methods

9 ,5

C e l l c u l t u r e s

E le c t r o n m ic ro s c o p y

T r a n s fo r m a t io n assay

O th e r i n v e s t i g a t i o n s

R e s u l ts

D is c u s s io n

Summary

Page

186186187187187188 192193

C h ap te r 10 GENERAL DISCUSSION AND THESIS FINDINGS

REFERENCES

APPENDICES A , R e s u l t s o f a b a t t o i r p a p i l lo m a s u rv e y

B, BPV p a r t i c l e d ia m e te r measurements

C, H a e m a to lo g ic a l r e s u l t s

D, D e t a i l e d r e s u l t s o f m u l t i p l e i n o c u l a t i o n s

o f p a s te u r is e d and c o n t r o l BPV i s o l a t e s

Eo BPV-in v i t r o t r a n s f o r m a t i o n assay r e s u l t s

194198243267297

508310

,7 .

Page

LIST OF TABLES

T a b le

1 The source of original isolates of papovaviruses 222 O n c o g e n ic i t y o f p o ly o m a v i ru s i n n o n -m u r in e s p e c ie s 263 O n c o g e n ic i t y of SV40 i n n o n -s im ia n s p e c ie s 264 Oncogenicity of BKV in non-human species 285 O n c o g e n ic i t y o f 3CV i n non-human s p e c ie s 2 9

6 P re v a le n c e o f v e r ru c a e i n S c o t t i s h s c h o o l e n t r a n t s 3 2

and le a v e r s f ro m 1970 to 1976

7 P a p o v a v iru s e s i n v i v o - h o s t range and o n c o g e n ic i t y 4-18 P r i n c i p a l c h a r a c t e r i s t i c s o f t ra n s fo rm e d c e l l s 439 C e l l s p e r m is s iv e f o r BKV r e p l i c a t i o n 4510 Cells that are BKV-transformation sensitive 4511 Papovavirus particle diameters 5812 Time-temperature inactivation of papovaviruses 6013 The p a p o v i r id a e i n v i v o g ]

14 The p a p o v i r id a e i n v i t r o gg

15 Im m u n o lo g ic a l and p h y s ic o - c h e m ic a l c h a r a c t e r i s t i c s 6 3

o f p a p o v a v i ru s e s

16 Number o f c a t t l e and t e a t s a f f e c t e d w i t h p a p i l l o m a t o s i s 7317 t - t e s t o f mean d i f f e r e n c e s i n % a n im a ls a f f e c t e d and

number o f a f f e c t e d t e a t s p e r a n im a l 7318 P r o p o r t i o n o f p a p i l l o m a ty p e s and number on a f f e c t e d

t e a t s 7419 Number o f p a p i l lo m a s o f each ty p e o c c u r r in g i n t h r e e

m u l t i p l i c i t y g ro u p in g s from a s e le c te d p o p u la t i o n 74.20 The d i s t r i b u t i o n o f t h r e e ty p e s o f b o v in e t e a t

p a p i l lo m a o c c u r r in g as one ty p e o r i n v a r io u s 77c o m b in a t io n s

21 Mean BPV p a r t i c l e d ia m e te rs measured on e l e c t r o n -

rn ic ro g ra p h s o f n e g a t i v e l y s t a in e d p u r i f i e d e x t r a c t s

o f v a r io u s p a p i l lo m a sam ples 8622 The deve lo p m e n t and r e g r e s s io n o f l e s io n s p roduced

at BPV-inoculation sites 9823 Challenge of inoculated calves by contact w i t h BPV

infected calves X0324 C h a l le n g e o f i n o c u la t e d c a lv e s by s c a r i f i c a t i o n

with BPV extracted from different lesion types 111

® 8 o

Page

T a b le

25 R e s u l t s o f s u b cu tan eous i n o c u l a t i o n o f BPV, BPV- 1 2 4

t r a n s fo r m e d FBSC and u n t r e a te d FBSC i n nude m ice

26 The r e s u l t o f i n v i v o t i t r a t i o n o f p a s te u r is e d and 1,44u n p a s te u r is e d B P V -c o n ta in in g e x t r a c t s on f i v e

c a lv e s

27 F o e ta l b o v in e t i s s u e s c o l l e c t e d , c e l l g ro w th p a s t ] g y

t h i r d passage and t r a n s f o r m a t i o n s e n s i t i v i t y to

BPV o f v a r io u s f o e t a l b o v in e c u l t u r e s

28 L o n g e v i t y o f p a i r e d t r a n s fo rm e d and c o n t r o l c u l t u r e s 1 6 0

29 The r e s u l t o f t r a n s f o r m a t i o n assay o f te n s e p a r a t e l y

p ro ce sse d f i b r o p a p i l l o m a s f ro m a s i n g l e case (65819 )

on th r e e d i f f e r e n t f o e t a l b o v in e c u l t u r e s * L6Q

30 The r e s u l t o f t r a n s f o r m a t i o n assay o f te n a l i q u o t s

o f p o o le d f i b r o p a p i l l o m a s f ro m a s i n g l e case (66702)

on th r e e d i f f e r e n t f o e t a l b o v in e c u l t u r e s 1 6 4

31 Time o f f i r s t appea rance o f t r a n s f o r m a t i o n w is p s i n

s u s p e n s io n and m on o la ye r i n f e c t e d c u l t u r e s I6432 The e f f e c t on t r a n s f o r m a t i o n - i n h i b i t i o n t i t r e o f

p r i o r serum a d s o r p t io n w i t h v a r io u s B P V - re la te d

s u s p e n s io n s 173

0 9 «

Page

LIST OF FIGURES

Figure1 The d i s t r i b u t i o n o f p a p i l lo m a s a c c o rd in g t o 7 6

m u l t i p l i c i t y p e r t e a t

2 T e a t p a p i l l o m a o f th e f ro n d , ty p e gg

3 T e a t p a p i l l o m a o f th e f l a t and round ty p e 69

4 T e a t p a p i l l o m a o f th e r i c e g r a in ty p e y g

5 E le c t r o n m ic r o g r a p h o f cu ta n e o u s f i b r o p a p i l l o m a

v i r u s 7 9

5 E le c t r o n m ic r o g r a p h o f t e a t f i b r o p a p i l l o m a v i r u s QQ

7 Electronmicrograph of teat rice grain papilloma 81virus

8 Electronmicrograph of teat papilloma virus 829 E le c t r o n m ic r o g r a p h o f dog cu ta n e o u s p a p i l lo m a

virus 8310 SDS-poly acrylamide gel of BPV extracts 8411 S D S -po ly a c r y la m id e g e l of BPV e x t r a c t s 85

12 H i s t o l o g i c a l appea rance o f t e a t r i c e g r a in l e s i o n 9713 H i s t o l o g i c a l appea rance o f t e a t p a p i l lo m a 10014 Histological appearance of teat fibropapilloma 10215 Histological appearance of cutaneous fibropapilloma 10616 Histological appearance of anogenital fibropapilloma 10717 Histological appearance of alimentary tract fibro

papilloma and papilloma 11318 M a c ro s c o p ic d i f f e r e n c e between t e a t and a n o g e n i t a l

f i b r o p a p i l l o m a s 11719 Nude mouse tum our g ro w th r e l a t e d to t im e p o s t -

i n o c u l a t i o n , 12520 Low-power p h o to m ic ro g ra p h s o f B P V - tra n s fo rm e d c e l l

in d u c e d nude mouse tum ours 1 2 7

21 High-power photomicrographs of in vitro transformedcells and nude mouse tumour cells 128

22 M a c ro s c o p ic and m ic ro s c o p ic appearance o f BPV-

induced meningioma 13623 The t im e - te m p e r a tu r e i n a c t i v a t i o n o f b o v in e

p a p i l lo m a v i r u s as measured by i n v i t r o t r a n s - 1 4 6

fo r m a t io n

24 B P V - t r a n s f o rm a t io n o f f o e t a l b o v in e s k in c u ltu res 154 -

10,

Page

F ig u re

25 The e f f e c t o f d i f f e r e n t a d s o r p t io n t im e s on th e

t r a n s f o r m a t i o n t i t r e o f b o v in e p a p i l lo m a v i r u s

infected foetal bovine skin cultures 15926 The e f f e c t o f b o v in e p a p i l lo m a v i r u s on th e g ro w th

curve of a foetal bovine skin culture 16227 BPV i n v i t r o t r a n s f o r m a t i o n i n h i b i t i o n assay t e s t

p l a t e 1 / 1

28 T r a n s fo r m a t io n i n h i b i t i o n t i t r e s o f e x p e r im e n ta l

c a l f se ra i n r e l a t i o n to p o s t - i n o c u l a t i o n t im e o f

deve lop m e n t and r e g r e s s io n o f f i b r o p a p i l l o m a s 1 7 6

29 The e f f e c t o f v a r y in g c o n c e n t r a t io n s o f v i r u s and

complement on t r a n s f o r m a t i o n i n h i b i t i o n u s in g one

h ig h t i t r e serum 1 7 8

30 T r a n s fo r m a t io n assay t e s t p l a t e 18031 Morphological changes seen in vitro when high

concentrations of virus and high titre transform- 181ation inhibition serum are used

32 E le c t r o n m ic r o g r a p h o f BPV p re s e n t i n c u l t u r e medium

24 days post-inoculation ^8933 R a d i o a c t i v i t y i n CsC1 g r a d ie n t s o f medium sam ples

f ro m B le o m y c in - t r e a te d and u n t r e a te d BPV i n f e c t e d

cell cultures 190

11.

LIST OF ABBREVIATIONS

( i n o r d e r o f f i r s t appearance i n th e t e x t )

nm nanom etres

mm m i l l i m e t r e s

cm c e n t im e t r e s

ml m i l l i l i t r e s

1 l i t r e s

m^ a p p a re n t m o le c u la r w e ig h t

% p e r c e n t

HPV human p a p i l lo m a v i r u s

BPV b o v in e p a p i l lo m a v i r u s

SV40 s im ia n v a c u o la t i n g v i r u s 40

EV e p id id e rm o d y s p l a s i a v e r r u c i f o r m i s

RKV r a b b i t k id n e y v a c u o la t i n g v i r u s

FPV f r i n g e l l a e p a p i l lo m a v i r u s

ROPV r a b b i t o r a l p a p i l lo m a v i r u s

SPV Shope p a p i l lo m a v i r u s

DPV o v in e p a p i l lo m a v i r u s

TSTA t u m o u r - s p e c i f i c t r a n s p l a n t a t i o n a n t ig e n

DNA d e o x y r ib o n u c le i c a c id

RNA r i b o n u c l e i c a c id

EM e l e c t r o n m ic ro s co p e

PML p r o g r e s s iv e m u l t i - f o c a l le u k o e n c e p h a lo p a th y

STMV s tu m p ta i l e d macaque v i r u s

UV u l t r a - v i o l e t l i g h t

PV p a p i l lo m a v i r u s

CPE c y t o p a t h i c e f f e c t s

RDE r e c e p t o r d e s t r o y in g enzyme

PHFG p r im a r y human f o e t a l g l i a l c e l l s

°C deg rees c e n t ig r a d e

CAM c h o r i o a l l a n t o i c membrane

VP v i r a l p r o t e i n

V - a n t ig e n v i r i o n a n t ig e n

T - a n t ig e n i n t r a n u c l e a r tum our a n t ig e n

IF im m u n o f lu o re s c e n t

CF complement f i x a t i o n

TSSA tum our a s s o c ia te d c e l l s u r fa c e a n t ig e n

C - a n t ig e n c y to p la s m ic a n t ig e n

TATA c e l l s u r fa c e s p e c i f i c g r a f t r e j e c t i o n a n t ig e n

>1 2 ,

ID im m u n o d i f fu s io n

SLE s y s te m ic lu p u s e t y th e m a to s is

DHR d e la y e d h y p e r s e n s i t i v i t y r e a c t i o n

CMI c e l l m e d ia te d im m u n i ty

LT ly m p h o c y te t r a n s f o r m a t i o n

LMIF le u k o c y te m ig r a t i o n i n h i b i t i o n f a c t o r

RBC re d b lo o d c e l l - e r y t h r o c y t e

BBC w h i t e b lo o d c e l l - le u k o c y te

HA h a e m a g g lu t in a t io n

HAI h a e m a g g lu t in a t io n i n h i b i t i o n

OMPU ow l monkey p a p o v a v i ru s

FEN f o c a l e p i t h e l i a l : h y p e r p la s ia

mg m i l l i g r a m

g gram

MCI h y d r o c h lo r i c a c id

SDS sod ium dodec y l s u lp h a te

V v o l t s

PTA p h o s p h o tu n g s t ic a c id

SD s ta n d a rd d e v i a t i o n

SE s ta n d a rd e r r o r

y g r e a t e r th a n

le s s th a n

CsC1 caes ium c h l o r i d e

TD t r a n s f o r m in g dose

TID t r a n s f o r m a t i o n i n h i b i t o r y dose

FBS f o e t a l b o v in e serum

BME b a s a l Eagle ’s medium

FBSC f o e t a l b o v in e s k in c e l l s

FBMC f o e t a l b o v in e m e n in g e a l c e l l s

T t r a n s fo r m e d

rpm r e v o l u t i o n s p e r m in u te

min m in u te

h r ho u r

log^Q l o g a r i t h m to base 10

NOTE A b b r e v ia t io n s s p e c i f i c t o i n d i v i d u a l c h a p te r s appea r i n

b r a c k e ts f o l l o w i n g th e f i r s t appearance o f each e x p r e s s io n

and a re n o t l i s t e d h e re .

13

ACKNOWLEDGEMENTS

I wou ld l i k e t o th a n k ;

; P r o fe s s o r W, F, H. GARRETT, my s u p e r v i s o r , f o r p e r m is s io n to use

th e f a c i l i t i e s and m a t e r i a l s needed f o r th e r e s e a r c h ; f o r making

t im e a v a i l a b l e t o g iv e a d v ic e , s u p p o r t and c o n s t r u c t i v e c r i t i c i s m

when c i r c u m s ta n c e s p e r m i t t e d ; and f o r th e a lm o s t superhuman p a t ie n c e

needed to read and im p rove t h i s t h e s i s and th e s c i e n t i f i c p a p e rs t h a t

r e s u l t f ro m i t ,

Î Mrs E, LEIGHTON has p ro v id e d h i g h l y s p e c ia l i s e d t e c h n i c a l s u p p o r t

i n th e p e r fo rm a n c e o f th e t r a n s f o r m a t i o n and t r a n s f o r m a t i o n i n h i b i t i o n

assays r e p o r t e d i n C ha p te rs 7 and 8 ,

: M r, Wo PENNY and s t a f f who p ro v id e d h i s t o p a t h o l o g i c a l s e c t io n s

o f t i s s u e s r e p o r t e d i n C ha p te rs 3 , 4 and 5,

; M rs , C, McLAY who p ro v id e d t h i n s e c t io n s as r e p o r t e d i n C hap te rs

2, 10 and e ls e w h e re i n t h i s t h e s i s ,

: Mr, At. FINNIE and s t a f f who p r o v id e d p h o to g ra p h s and p h o to m ic ro

g raphs o f tum ours r e p o r t e d th ro u g h o u t th e t h e s i s ,

; M r, 3 , MORRISON and Mr, C^ W i ls o n who p ro v id e d th e e l e c t r o n -

m ic ro g ra p h s i n C h a p te rs 2 and 9 ,

: Mr* 3 . MURPHY and s t a f f f o r a s s is ta n c e i n th e c o n d u c t o f b o v in e

p o s t-m o r te m e x a m in a t io n s ,

: M r, 0 . McINNES f o r husban d ry o f th e c a t t l e r e p o r t e d i n C ha p te rs

3, 5 and 6»

; M r, A, BRADLEY, M rs , M. BOAG and s t a f f f o r th e husban d ry o f th e

e x p e r im e n ta l a n im a ls r e p o r te d i n C h a p te r 4 ,

; P r o fe s s o r D, LAWSON and s t a f f who p e r fo rm e d th e i n t r a c e r e b r a l

i n o c u l a t i o n s r e p o r te d i n C h a p te r 5*

s D r , Do 3ARRETT and s t a f f who p ro v id e d e x p e r t a d v ic e on th e

c u l t u r a l a n d v i r o l o g i c a l te c h n iq u e s r e p o r te d i n C h a p te rs 7 , 8 and 9 ,

: M rs , M, McCREADIE, M rs , M, TROY and M rs , H* ZIKAKIS who t o g e th e r

have ty p e d t h i s t h e s i s ,

: D r , 1 , McCANDLISH who a s s is t e d w i t h th e lu n g h i s t o p a t h o l o g y *

; Mr, A, SPENCER who a s s is t e d w i t h th e k id n e y h i s t o p a t h o lo g y ,

: M rs , P, ROGERSON who a s s is t e d w i t h th e l y m p h o r e t i c u la r

h i s t o p a t h o l o g y ,

: M r, B, CHNEIL f o r a s s is ta n c e i n e n s u r in g th e c o n s ta n t f l o w o f th e

n e ce ss a ry m a t e r i a l s r e p o r te d th r o u g h o u t t h i s t h e s i s ,

: D r , Do ONIONS who a s s is t e d w i t h im m u n o lo g ic a l a d v ic e *

; M iss A, McRAE who p e r fo rm e d h a e m a to lo g ic a l e x a m in a t io n s ,. 1 4 .

: Mr* 3o NEIL who p e r fo rm e d th e p o ly a c r y la m id e g e l e l e c t r o p h o r e s i s

te c h n iq u e s r e p o r t e d i n C h a p te r 2 , and has p ro ve d a v a lu a b le s ou rce

o f a d v ic e on v i r o l o g i c a l a s p e c ts o f th e w o rk .

; M r. C«Mo McCANCE o f th e Glasgow C i t y A b a t t o i r , f o r p e rm is s io n

t o c a r r y o u t th e s u rv e y r e p o r te d i n C h ap te r 2 .

; D r . H, LAIRD, who s p e n t much v a lu a b le t im e te a c h in g me e le c t r o n

m ic ro s c o p y , and s u b s e q u e n t ly g i v i n g a d v ic e on a s p e c ts o f th e u l t r a -

s t r u c t u r a l work r e p o r t e d i n t h i s th e s is ®

: The A u s t r a l i a n Meat Research C om m ittee ; th e Commonwealth

Government o f A u s t r a l i a and th e A g r i c u l t u r a l R esearch C o u n c i l o f

G re a t B r i t a i n who p ro v id e d f i n a n c i a l a s s is ta n c e w h ich i s g r a t e f u l l y

acknowledged®

; Dr® M*A*S® JONES, e x e c u t i v e o f f i c e r o f th e A u s t r a l i a n Meat

Research Com m ittee and Dr® R®W* GEE, d i r e c t o r o f th e A u s t r a l i a n

Bureau o f A n im a l H e a l th who have g iv e n enormous encouragem ent and

s u p p o r t *

I am in d e e d f o r t u n a t e i n h a v in g a p a t i e n t and u n d e rs ta n d in g

w i f e , who, t o g e t h e r w i t h t h r e e c h i l d r e n s u f f e r e d c o n s id e r a b ly t o

a l lo w t h i s work t o p roceed t o f r u i t i o n ® T h e i r s u p p o r t was o f

c r i t i c a l im p o r ta n c e and i s acknow ledged w i t h g r a t i t u d e ®

H® R, C® MEISCHKE

S eptem ber, 1978®

o 15«

DECLARATION

I h e re b y d e c la r e t h a t t h i s t h e s i s embodies th e r e s u l t s o f my own

s p e c ia l w o rk , t h a t i t has been composed by m y s e l f and t h a t i t does n o t

i n c lu d e work fo r m in g p a r t o f a t h e s i s p re s e n te d s u c c e s s f u l l y f o r a

degree i n Glasgow U n i v e r s i t y o r a n o th e r U n i v e r s i t y .

S ig n a tu r e

Date

.1 6 .

SUMMARY

An a b a t t o i r s u rv e y r e p o r t e d an o v e r a l l t e a t p a p i l l o m a t o s i s p re v a le n c e

o f 36 '% i n 721 c a t t l e exam ined . A f f e c t e d a n im a ls possessed an a ve rage o f

f i v e p a p i l lo m a s on each o f two a f f e c t e d t e a t s . S i g n i f i c a n t sax d i f f e r

ences o c c u r re d i n th e p r e v a le n c e o f th e d is e a s e , and n u l l i p a r o u s

fe m a le c a t t l e had s i g n i f i c a n t l y fe w e r a f f e c t e d t e a t s th a n pa rous

fe m a le s . M o r p h o lo g i c a l l y s e p a ra b le l e s io n s were seen and on h i s t o l o g i c a l

e x a m in a t io n were shown to be p a p i l lo m a s , f i b r o p a p i l l o m a s and f o c a l e p i t h

e l i a l h y p e r p l a s i a - l i k e l e s i o n s . B ov ine p a p i l lo m a v i r u s was e x t r a c te d

f ro m a l l t h r e e l e s i o n t y p e s . E le c t r o n m ic r o s c o p ic e x a m in a t io n re v e a le d

t h a t BPV e x t r a c t e d f ro m t e a t p a p i l l o m a , f i b r o p a p i l l o m a and f o c a l

e p i t h i a l h y p e r p l a s t i c l e s i o n had s i g n i f i c a n t l y d i f f e r e n t p a r t i c l e

d ia m e te r s . P o ly a c r y la m id e g e l e l e c t r o p h o r e s i s o f BPV fro m cu taneo us

f i b r o p a p i l l o m a s showed a m a jo r c a p s id p r o t e i n band w h ic h has a c o n s i s t e n t l y

h ig h e r a p p a re n t m o le c u la r w e ig h t th a n t e a t l e s i o n ' e x t r a c t s . E p id e m io l

o g i c a l e v id e n c e s u g g e s te d th e p re se n ce o f a t l e a s t two s e p a ra te v i r u s /

h o s t i n t e r a c t i o n s were p re s e n t s in c e th e d i s t r i b u t i o n s o f th e i n d i v i d u a l

t e a t l e s i o n s d i f f e r e d i n b o th number and shape o f c u r v e .

When s i n g l e ca se , s i n g l e l e s i o n ty p e BPV e x t r a c t s were in o c u la t e d

i n t o e x p e r im e n ta l c a l v e s , f o u r m o r p h o lo g ic a l l y s e p a ra b le l e s i o n s r e s u l t e d .

BPV f ro m t e a t and cu ta n e o u s f i b r o p a p i l l o m a s p roduced cu ta n e o u s f i b r o

p a p i l lo m a s w h ich were m o r p h o lo g i c a l l y i n d i s t i n g u i s h a b l e f ro m each o t h e r ;

BPV f ro m a n o g e n i t a l f i b r o p a p i l l o m a s p roduced c u taneo us f i b r o p a p i l l o m a s

m o r p h o lo g i c a l l y s e p a ra b le f ro m th o s e above; BPV f ro m t e a t p a p i l lo m a and

f o c a l e p i t h e l i a l h y p e r p l a s t i c l e s io n s p roduced p a p i l lo m a and f o c a l

e p i t h e l i a l h y p e r p l a s t i c le s io n s r e s p e c t i v e l y on t r a n s m is s io n to no rm a l

b o v in e s k i n . BPV was e x t r a c t e d f ro m a l l t r a n s m i t t e d le s io n s ®

E x p e r im e n ta l c a lv e s were c h a l le n g e d w i t h BPV f ro m a l l f i v e l e s i o n

ty p e s and a d i f f e r e n t i a l im m u n i ty was dem onstra ted® The f i b r o p a p i l l o m a

c a lv e s were immune to c h a l le n g e w i t h most b u t n o t a l l f i b r o p a p i l l o m a

i s o l a t e s , b u t s u s c e p t i b l e t o c h a l le n g e w i t h t e a t p a p i l l o m a and f o c a l

e p i t h e l i a l h y p e r p la s ia i s o l a t e s . C o n v e rs e ly p a p i l l o m a and f o c a l

e p i t h e l i a l h y p e r p la s ia BPV i n f e c t e d c a lv e s rem a ined s u s c e p t i b l e t o a l l

f i b r o p a p i l l o m a i s o l a t e s and r e g r e s s io n o f f i b r o p a p i l l o m a s was n o t accom

p a n ie d by th e r e g r e s s io n o f th e fo rm e r le s io n s ® I t was consWered t h a t

two b road v i r u s c a t e g o r ie s o c c u r re d w i t h p a p i l lo m a and f o c a l e p i t h e l i a l

h y p e r p la s ia BPV fo r m in g one and f i b r o p a p i l l o m a BPV th e o t h e r c a te g o r y .

W i t h in th e s e two c a te g o r ie s t h e r e were m o r p h o lo g ic a l and d e v e lo p m e n ta l

d i f f e r e n c e s s u g g e s t in g f u r t h e r h e te r o g e n e i t y w h ich may be c o n f i rm e d .

w i t h th e use o f l a r g e r numbers o f e x p e r im e n ta l a n im a ls *

.1 7 .

When i n o c u la t e d i n t r a c e r e b r a l l y , two f i b r o p a p i l l o m a BPV i s o l a t e s

p roduced meningiomas® One i s o l a t e was from a s i n g l e case o f cu taneous

f i b r o p a p i l l o m a s . The o th e r was fro m cu taneous f i b r o p a p i l l o m a s w h ich

appea red a lo n g s c a r i f i c a t i o n l i n e s o f a c a l f e x p e r im e n t a l l y i n o c u la te d

w i t h BPV e x t r a c t e d f ro m a p o o le d sample o f a l im e n t a r y l e s i o n s . A

t h i r d c a l f , i n t r a c e r e b r a l l y i n o c u la t e d w i t h BPV fro m s i n g l e case t e a t

f o c a l e p i t h e l i a l h y p e r p l a s t i c l e s i o n s , deve lop ed s i m i l a r l e s io n s a t

i n t r a d e r m a l s i t e s b u t d id n o t d e v e lo p a m en ing iom a.

In v i t r o , f o e t a l b o v in e s k i n , c o n ju n c t i v a , p a la t e and meninges

d e r iv e d c e l l c u l t u r e s were shown to be s e n s i t i v e t o t r a n s f o r m a t i o n by

BPV e x t r a c t e d fro m f i b r o p a p i l l o m a s b u t n o t p a p i l lo m a s and f o c a l

e p i t h e l i a l h y p e r p l a s t i c le s io n s ® O n ly two s k in c u l t u r e s were s e n s i t i v e

and b o th o f th e s e came from n e a r - te r m fo e tu s e s . C u l t u r e s f ro m th e p ia

m a te r and a ra c h n o id e a were s e n s i t i v e w h i le du ra m a te r d e r iv e d c u l t u r e s

were n o t . The i n v i t r o t r a n s f o r m a t i o n assay was d eve lop ed and shown

t o be more s e n s i t i v e and p r e c is e th a n h a e m a g g lu t in a t io n , however th e two

system s p ro b a b ly measure d i f f e r e n t p a ra m e te rs . No s i g n i f i c a n t d i f f e r e n c e s

o c c u r re d i n th e t r a n s f o r m a t i o n t i t r e o f two BPV i s o l a t e s when d i f f e r e n t

t i s s u e s o f t h e same fo e tu s and th e same t i s s u e s o f d i f f e r e n t fo e tu s e s

were used ,

B P V - tra n s fo rm e d c e l l s were shown to be tum ourigen ic i n nude m ice

b u t n o t i n c a lv e s f o l l o w i n g i n t r a c e r e b r a l , s u b cu ta n e o u s , in t r a d e r m a l

and subm ucosa l i n j e c t i o n . However, i n o c u la t e d c a lv e s showed an in c re a s e d

i n v i v o r e s i s t a n c e to c h a l le n g e w i t h f i b r o p a p i l l o m a d e r iv e d BPV b u t n o t

p a p i l lo m a o r f o c a l e p i t h e l i a l h y p e r p la s ia d e r iv e d BPV, B P V -tra n s fo rm e d

c e l l s showed an in c re a s e d l i f e s p a n when compared w i t h c o n t r o l c u l t u r e s .

The m o r p h o lo g ic a l changes in d u c e d by BPV were h e r i t a b l e i n a l l c u l t u r e s

and, w i t h one e x c e p t io n , p e r s i s t e d th ro u g h o u t th e i n v i t r o l i f e s p a n o f

each c u l t u r e . One f o e t a l s k in c u l t u r e showed a r e v e r s io n t o no rm a l

m o rpho log y a p p r o x im a te ly s i x months f o l l o w i n g i n f e c t i o n by BPV. The r e v -

e r t a n t c e l l s were r e s i s t a n t t o r e - t r a n s f o r m a t i o n by BPV even though

p a i r e d c o n t r o l c u l t u r e s rem a ined t r a n s f o r m a t i o n - s e n s i t i v e .

In v i t r o t r a n s f o r m a t io n by BPV c o u ld be i n h i b i t e d by s e ra f rom

f i b r o p a p i l l o m a b e a r in g c a lv e s . T r a n s f o r m a t i o n - i n h i b i t i o n ( T I ) was

shown t o be complement dependen t b u t v i r u s in d e p e n d e n t o v e r a w ide range ,

o f d i l u t i o n s , , T I t i t r e s f i r s t appeared i n s e ra f ro m c a lv e s w i t h f i b r o - .

p a p i l lo m a s c o n c u r r e n t l y w i t h th e f i r s t appearance i n b io p s y samples

o f c e l l m e d ia te d r e g r e s s io n as e v id e n ce d by ly m p h o c y te a c c u m u la t io n

i n th e f ib r o m a component. T I t i t r e s p e r s i s t e d f o r 4 - 8 weeks

f o l l o w i n g c o m p le te r e g r e s s io n o f f i b r o p a p i l l o m a s . I n BPV - in d u ce d

,1 8 .

m en ing iom as, no h i s t o l o g i c a l e v id e n c e o f r e j e c t i o n c o u ld be seen. T I

t i t r e s c o u ld n o t be d e te c te d i n serum samples f o l l o w i n g c om p le te

r e j e c t i o n o f c u ta n e o u s f i b r o p a p i l l o m a s on th e same c a lv e s d e s p i t e th e

p resence o f p r o g r e s s i v e l y d e v e lo p in g m en ing iom as. Meningiomas

appeared to o r i g i n a t e i n th e p ia m a te r s in c e i n b o th c a lv e s , th e du ra

m a te r was u n in v o lv e d , T I a n t ib o d y a c t i v i t y c o u ld be adso rbed from

h ig h t i t r e serum u s in g f ib r o m a , f i b r o p a p i l l o m a and m en ing iom a t i s s u e

su s p e n s io n s as w e l l as i n v i t r o t r a n s fo rm e d c e l ls ® T I a n t ib o d y

a c t i v i t y was n o t reduced f o l l o w i n g a b s o r p t io n w i t h p a p i l l o m a , f o c a l

e p i t h e l i a l h y p e r p l a s t i c , no rm a l b o v in e and f o e t a l b o v in e t i s s u e

s u s p e n s io n s , f o e t a l b o v in e c e l l c u l t u r e s , as w e l l as a l l c l a r i f i e d and

p u r i f i e d BPU s u s p e n s io n s , w i t h one exce p t io n ® The cu ta n e o u s f i b r o

p a p i l lo m a d e r iv e d p u r i f i e d v i r u s su s p e n s io n c o n ta in e d s u f f i c i e n t BPV

t o o v e r - r i d e th e no rm a l v i r u s c o n c e n t r a t i o n indepe ndence o f th e assay

system»

BPV was shown to be r e s i s t a n t t o p a s te u r is a t io n i n v i v o u s in g

s e p a ra te e x t r a c t s o f a l l th e m a jo r l e s i o n typ e s» In v i t r o , f i b r o

p a p i l lo m a d e r iv e d BPV showed a t e n - f o l d f a l l i n t r a n s f o r m a t i o n t i t r e

f o l l o w i n g 4 h o u rs a t 60°C and a l l t r a n s f o r m a t i o n a c t i v i t y ceased a f t e r

30 m in u te s a t 8G°Co BPV was d e te c te d i n two o f s i x m i l k samples

pu rchased a t r e t a i l o u t l e t s . The l i k e l y w id e s p re a d e x p o s u re o f humans

t o BPV i n m i l k , c o u p le d w i t h a s i m i l a r i t y between some t e a t l e s io n s and

o r a l human l e s io n s le d to th e s u g g e s t io n t h a t th e q u e s t io n o f w h e th e r

BPV can i n f e c t man be i n v e s t i g a t e d f u r t h e r .

O n ly 3 o f 44 c u l t u r e s t e s te d f o r i n v i t r o BPV r e p l i c a t i o n showed

BPV i n th e medium. T h is o c c u r re d between 24 - 28 days p o s t i n f e c t i o n ®

R a d i o - l a b e l l i n g w i t h t r i t i a t e d th y m id in e showed th e p resence o f a peak

i n BPV c o n t a in in g medium p u r i f i e d i n CsCI. . The r e s u l t s o f r a d i o

l a b e l l i n g , b u o ya n t d e n s i t y and e l e c t r o n m ic ro s c o p y su g g e s te d t h a t BPV

had been c u l t i v a t e d i n v i t r o . The r e s u l t s ware f a r f ro m c o n c lu s i v e , b u t

th e c o m b in a t io n o f p ro lo n g e d c o n t a c t i n h i b i t i o n and s e v e re s t r e s s may

have c o n t r i b u t e d to th e phenomena o b s e rv e d .

o 1 9 ®

c J ]a p te r i . c e n t r a i in t r o d u c t io n a n d l i t e r a t u r e r e v ie w

"W lien v iru s e s in te ra c t w ith ce lls they b r in g about a lte ra tio n s in the

DNA content o f the c e lls . Though the p re c ise m echan ism s m ay be d if fe re n t

th is is a lso tru e o f ch em ica l carc inogens and ra d ia tio n . The end re s u lt is

de te rm ined p a r t ly b y the natu re of the damage but a lso in p a r t by the capac ity

o f the c e ll to re p a ir the damage. These events m ay p la y an im p o rta n t p a rt

in the in it ia t io n o f neop lasia , ” (Harnden, 1976).

1.1 The Papovaviridae

T lie te rm papovav irus was f i r s t used by M e in ic k (1962) to denote a

group o f v iru s e s w ith com m on c h a ra c te r is t ic s . P ap illom a , po lyom a and

va cuo la ting agent (SV40) each p ro v id e th e ir f i r s t tw o le t te rs to fo rm the g roup

name,Papova. The In te rn a tio n a l C om m iss ion fo r the N om enc la tu re o f V iru s e s

o f f ic ia l ly approved the name Papovaviridae w h ich is now used to denote a

fa m ily o f v iru s e s tha t is subd iv ided in to tw o genera, p a p illo m a v iru s and

p o ly o m a v iru s , w ith ra b b it (Shope) p a p illo m a v iru s and p o lyo m a v iru s the

re sp ec tive type species.

1.1.1 G enera l P ro p e rties o f Papovaviruses

The v iru s e s a re 40-57 nm in d ia m e te r and, as d e te rm ine d by negative

s ta in ing , the o u te r s h e ll has a s y m m e try of the T ™ 7 ico sa he d ra l su rface

la t t ic e and is composed of 72 m o rp h o lo g ica l subunits (F inch and K lig 1965;

A n d e re r e t a l. , 1967). The v iru s e s a re e the r stab le (conta inno essen tia l l ip id s ) .

They conta in DNA, p re se n t as a c o va le n tly closed dup lex m o lecu le , w ith in the

co re of the v iru s p a r t ic le . P a p illo m a v iru se s have DNA o f a m o le c u la r w e ight

c lose to 5 X 10^ da ltons, h ig h e r than the m o le cu la r w e ights o f SV40 (3. 6 x 10^)

(T a i et a l. , 1972) and po lyom a (3 .0 x 10^) (W eil and V inog rad , 1963). The

D N A ’s o f the papovav iruses a re in fec tio u s and are able to tra n s fo rm c e lls in

v i t r o (M cCutchan and Pagano, 1968; C ra w fo rd et a l. , 1964; Bourgaux et a l. ,

1969; A aronson and T odaro , 1969; Aaronson and M a rtin , 1970).

1.1. 2 O r ig in a l Iso la tio n o f the Papovaviruses

Tab le 1 l is ts the known m em bers o f the papovav iridae and th e ir source

o f o r ig in a l is o la tio n . A t the p o in t of w r it in g , the num ber o f se p a ra te ly

20

id e n tif ie d papovaviruses stands at tw en ty-seven . Evidence fo r existence

of se ve ra l su bs tra in s w ith in recogn ised types is m ounting as w e ll as the

re la tedness o f se pa ra te ly recogn ised types.

T h ree re c e n tly iso la ted s tra in s o f JCV, MAD 1, MAD 2 and

M AD 4 have shown a d if fe re n t ia l neu rooncogen ic ity fo llo w in g in tra c e re b ra l

in ocu la tion in new born h a m s te rs , MAD 1 and MAD 2 produced m edu llo -

b lastom as of the ce re be llu m w h ile M AD 4 tended to p roduce p inea l gland

tum ou rs (Padgett e t a l. , 1977). W ith in the p a p illo m a v iru s g roup , the

ex is tence o f separa te v iru s e s causing les ions re s tr ic te d to c e r ta in anatom ica l

areas and tissues has been known fo r a long tim e . The ra b b it has both a skin

p a p illo m a v iru s and an o ra l p a p illo m a v iru s as does the dog. M ore re c e n tly ,

the exis tence of s e ve ra l d if fe re n t human p ap illom a v iru s e s (HPV) has been

re p o rte d . V iru s fro m g en ita l and la ryn ge a l pap illom as , and v iru s fro m

ep ide rm odysp las ia v e r ru c ifo rm is (EV) have been shown to be d if fe re n t f ro m

v iru s e x trac te d fro m the com m on sk in w a rt. (A lm e ida et a l , , 1969; Zu rliausen

et a l. , 1974; O rth e t a l . , 1978,* G is s man et a l. , 1977,* O rth e t a l , , 1977),

T h is thes is w i l l con ta in evidence fo r the existence o f se v e ra l types o f bovine

pap illom a v iru s (BPV) (M eischke, 1978<L-a),

Severa l types o f po lyom av iruses have been com pared im m u n o lo g ica lly ,

b io ch e m ic a lly , in v iv o and in v i t r o . The re s u lts show v a ry in g degrees of

re la tedness between m em bers o f the group. C ross re a c tio n s among the

V antigens of SV40, BKV, JCV and RFV have been observed b y haem agg lu tina tion

in h ib itio n (HI) (G a rd n e r c t a l. , 1971; W a lke r e t a l. , 1974),n e u tra lis a tio n

(B ernhard t c t a l . , 1975; D ougherty and d i Stefano, 1974), f lu o re sce n t s ta in ing

(Takem oto and M u lla rk e y , 1973) and im m une agg lu tina tion (A lb e rt and Zu T he in ,

1974; F ie ld e t a l . , 1974; G ardne r et a l. , 1971; Penny and N arayan, 1973).

C ross re a c tio n s among the non-V antigens have a lso been observed . The

in tra n u c le a r T antigen has been shown to be v iru s re la te d and not host c e ll

re la te d , T iie n uc lea r T antigens of SV40, R FV, BKV and JCV have been

shown to be s im i la r w ith s tro ng c ross re a c tion s observed b y flu o re sce n t

s ta in ing , w h ile po lyom a T antigen is d iffe re n t fro m a ll fo u r (Shah et a l . , 1973;

Takem oto and M u lla rk e y , 1973; W a lke r et a l . , 1973). T lie v iru s induced

tum ou r c e ll antigen, U , is a lso c ro s s re a c tiv e w ith a n ti SV40 U an ti se rum

re a c tin g w ith BKV and JCV in fec ted c e lls (Takem oto, 1975 pe rsona l

com m un ica tion , c ited by Padgett and W a lke r, 1976), The v iru s sp e c ific

. 21 .

TABLE L THE SOURCE OF ORIGINAL ISOLATES OF PAPOVAVIRUSES

VIRUS SOURCE OF O R IG IN AL ISOLATION REFERENCE

Polyoma p a ro tid gland tum ou rs of mouse G ross (1953), S tew art (1953).

SV40 m onkey c e ll cu ltu re s Sweet and PI f ile man (1960).

BKV u rin e o f rena l tra n sp la n t pa tien t m onkey c e ll c u ltu re s ,hum an foe ta l k idney, lung, b ra in cu ltu re s

re v ie w by Padgett and W a lke r &976X

JCV human b ra in w ith p ro g re s s iv e m u lt ifo c a l leukoenc eph a 1 op a th y Padgett et a l . , (1971).

R FV u rin e of re n a l tra n sp la n t pa tien t D ougherty (1976).

M M V re t ic u lu m c e ll sa rcom a of b ra in and u rin e Takem oto et a l . , o f p a tie n t w ith W is k o tt-A ld r ic h syndrom e (1974).

STM V s tum p ta iled m onkey k idney cu ltu re s Rangan et a l . , (1974).

HD - m onkey k idney c e ll cu ltu re s Wal .deck & Sauer (1977).

K. v iru s RKV

re s p ira to ry t ra c t and faeces of m ice ra b b it k idney c e ll cu ltu re s

re v ie w by Andrewes and P e re ira (1972)

unnamedpapovav iru s e s

sw ine papovav irus

lym phocytes o f m easles patien ts b ra in of p a tien t w ith C reu tz fe ld t-Jakob d iseaseb ladde r e p ith e liu m of pa tien t w ith acute h a e m o rrh a g lc c y s t it is of childhood (APIC) k idney c e ll cu ltu re s o f the ow l m onkey

Newman and Sm ith (1972)Le cats a 8 et a l . , (1976) deReuck et a l . , (1976)

Has h id a e t a l. (1976).

D an ie l et a l . , (1976). .

2 2 .

TABLE l .(C o n t’d ).T H E SOURCE OF ORIGINAL ISOLATES OF PAPOVAVIRUSES

BPV

HPV

VIRUS A N IM A L TUM OUR REFERENCE

FPV cha ffin ch sk in p ap illom as Ostex'haus et a l . , 1977.opossum sk in p ap illom as K o lle r , 1972.

Shope ra b b it sk in p ap illom as and Shope, 1933.ca rc inom as

ROPV ra b b it o ra l p ap illom as Parsons and K idd , 1943.dog sk in pap illom as W atrach , 1969.dog o ra l pap illom as C ham bers and Evans, 1959,chim panzee fo ca l e p ith e lia l O rth , 1976.

h yp e rp la s ia

OPV sheep sk in fib ro p a p illo m a s Head, 1965,goat sk in pap illom as D av is and K anper, 1936,goat udder p ap illom as and

ca rc inom as M ou lton , 1954d ee r sk in f ib ro m a s ;f ib ro

p ap illom as Shope e t a l , , 1958.

G ra f f i h a m s te r sk in pap illom as G ra f f i e t a l , , 1970,p ig g e n ita l p ap illom a Papish, 1961.m onkeys sk in p ap illom as See sec tion 1, 3,17.

c a ttleca ttle

c a t t leca ttleca ttle

human

s k in f ib r opap i l l om a s anogen ita l f ib ro - p ap illom as sk in p ap illom as a lim e n ta ry pap illom as tea t p a p illo m a s , f ib r o p a p illo m a s and r ic e - g ra in les ions

sk in p ap illom as fo ca l e p ith e lia l h yp e rp la s ia anogen ita l and o ro - la ryn g e a l p ap illom as

C reech , 1929. M cE ntee, 1952.

B artho ld e t a l. , 1974. J a r re tt e t a l. , 1978, M e ischke, 1978.

C u iffo , 1907,P ra e to riu s -C la u se n , 1972.

Rows on and M ahy, 1967.

. 23 .

tu m o u r-s p e c if ic tra n sp la n ta tio n antigen (TSTA) dem onstra ted b y p ro te c tive

e xpe rim en ts in h a m s te rs , how ever, appears not to c ross re a c t between

JCV, BKV and SV40 induced tu m o u r c e lls (Padgett e t a l . , 1977; Padgett and

W a lke r 1976). W liile v iru s p ro te in s of SV40, BKV and JCV are antigenic a lly

re la te d , the m a jo r p ro te in o f BKV is s m a lle r than tha t o f SV40 but the m in o r

p ro te in s have s im i la r m o le c u la r w e ights (M u lla rlcey et a l. , 1974; W alker

et a l. , 1974), U sing io d ina tio n o r t r y p t ic d igestion , som e o f the

polyiêptides o f SV40 and BKV showed d iffe re n ces in sp ite o f s im i la r m o le cu la r

w eights (W eiss e t a l . , 1975). An 11 - 12% hom ology in the " la te " re g io n of

SV40 DNA was found between the DNA of SV40 and BKV (K hour y et a l. , 1975).

F ro m the above exam ples, i t can be seen both tha t the re a re in te r

re la tio n s h ip s between d if fe re n t m em bers o f the papovav iridae and m arked

he te rogene ity between d if fe re n t iso la tes o f the same papovav irus . F o rm e r

c le a r ly defined d iffe re n ce s , s t i l l lis te d in c u rre n t textbooks, between the

p o ly o m a -v iru s e s and the p a p illo m a v iru s e s a re now le ss d is t in c t w ith

cons ide rab le ove rlap between the two groups in m o s t p a ra m e te rs . Polyom a

and SV40 are two of the m ost in te n s iv e ly studied v iru s e s in h is to ry^ fo r

exam ple the com plete nuc leo tide sequence of SV40 DNA has been published

(F ie rs e t a l . , 1978). There a re in excess of 1000 re fe re n ce s concern ing the

polyom a group of v iru s e s w h ich have been published s ince 1973, T h is thes is

w i l l concentra te on com para tive aspects o f the papovaviruses w hich bea r

d ire c t ly on p ro p e rtie s d isp layed by bovine p a p illo m a v iru s (BPV),

1.2 rolyo.ravirnuGU in Vivo - host range and oncogenicity.

T lie p o lyo m a v iru s group of the papovaviridae d is p la y a w ide range o f

oncogen ic ity in v iv o . Some m em bers a re nononcogenic w h ile o the rs are

oncogenic in a num ber o f an im a l spec ies. In gene ra l, those oncogenic

m em bers produce adenomas, ca rc inom as and fib ro s a rc o m a s o r a m ix tu re

of these tum ou rs in th e ir n a tu ra l host and o the r spec ies. T lie type o f tum ou rs

produced v a r ie s w ith the ro u te o f inocu la tion , the re la t iv e oncogen ic ity o f the

v iru s and the s u s c e p t ib ility o f the a n im a l. How ever, w ith ra re exceptions,

the v iru s e s produce tum ou rs re s tr ic te d to e p ith e lia l and m esenchym al tissu es

and th e ir equ iva lents w ith in the ce n tra l nervous sys tem . A lthough the re a re

a g re a t m any in te rre la tio n s h ip s between in d iv id u a l m em bers of the group , the

fo llo w in g pages w i l l a ttem p t to con fo rm to the m ost re c e n tly re p o rte d

. 2 4 .

sub "g roup ings of the p o lyo m a v iru se s ,

1 ,2 . 1 P o lyom av irus

A lthough neonates and c e rta in s tra in s o f m ice a re h ig h ly susceptib le

to po lyom a induced neop las ia (G ross, 1970; Rowe et a l . , 1962), g e n e ra lly the

v iru s is la te n t w ith tu m o u r in d uc tio n in outbred and in b red s tra in s a v e ry

ra re occu rrence (G ross, 1970). The wide range o f tum ou rs induced by

po lyom a v iru s in the mouse inc ludes:

sa rcom as - s a liv a ry glands; mucous and se rous g lands of the

head and neck; k idney; subcutaneous tissu e ;

bone and c a rtila g e .

ca rc inom as - s a liv a ry glands, mucous and serous g lands of

the head and neck; h a ir fo l l ic le ; ad rena l

m edu lla .

adenomas - s a liv a ry glands; mucous and serous glands of

the head and neck,

a lso “ adenocarcinom as of m a m m a ry gland,

m eso the liom as, endothe liom as, e p ith e lia l

th ym om as, haem angioendothe liom as.

H ow ever, the m ost com m on d isease induced by po lyom a in the mouse

is b ila te ra l, m u ltilo b u la te d neoplasm s o f the p a ro tid g land, (G ross, 1970;

S tew art, 1960).

P o lyom av irus is a lso oncogenic in o the r species. Table 2 l is ts the

species and the tjqês o f tum ou rs produced by p o lyo m a v iru s .

Polyom a produces p r im a r i ly ca rc inom as and sa rcom as o f a w ide range

o f tissues and organs in its n a tu ra l host w h ile the m esenchym al tu m o u rs ,

sa rcom a and fib ro m a a re produced fo llo w in g e xp e rim e n ta l tra n s m is s io n to

o the r species.

25

TABLE 2: ONCOGENICITY OF POLYOMAVIRUS IN NON-MURINE SPECIES

Species T u m o u r induced References

h am ste rs

new born h a m s te r

ra t

m astom ys

fe r re t

guinea p ig

ra b b it

sarcom a

m eningeal sarcom a

sarcom a

sarcom a

sarcom a

sarcom a

fib ro m a

Eddy et a l. , (1958)Berm an (1967)

Rabson and K irs c h s te in (1960)

Eddy et a l . , (1959)a

Rabson et a l. , (1960)

H a r r is e t a l. , (1961)

Eddy et a l. , (1960)

Eddy et a l . , (1959)

1 .2 .2 SV40

As w ith po lyom a, SV40 produces a la te n t in fe c tio n in Rhesus,

6ynom ol gus and 6*eropithecus m onkeys (Eddy et a l , , 1962; Sweet and H ille m an ,

1960). H ow ever, when g iven to suc ld ing g r iv e ts , baboons orRrhesus m onkeys,

SV40 produces f ib ro s a rc o m a ta . (A ndrew es and P e re ira , 1972).

SV40 is oncogenic in o th e r species. Table 3 l is ts the species and

types o f tum ou rs produced by SV40.

TABLE 3: O N C O G EN IC ITY OF SV40 IN N O N -S IM IAN SPECIES

Species T u m o u r induced R eferences

ham ste rnev/born

w eanling

choro id p lexus | K irs c h s te in and G e rb e r,(1962)papillom aêpendymoma^ I U n te rha rn sch e id t e t a l. , (1964) sa rcom a, fib rosa rcom a^ Berm an, (196'^; Eddy, (196^;

D u ffe l e t a l , , fl96'ÿ.

Mastom ys

lym p h ocy tic leukem ia ,1 ym phosarc om a, re tic u lu m c e ll sarcom a,! osteogenic sarcom a^ j anap las tic sarcom as of | v a rio u s organs |

ependym om a

D iam and opoulos, (l973).

Rabson et a l . , (l962).

.26.

SV 40 has been a c c id e n ta lly inocu la ted in to la rg e iiu in b e rs o f people as a

re s u lt o f con tam ina tion of tissue c u ltu re d e rived p o lio m y e lit is vacc ine . A

fu l l d iscuss ion and re v ie w on th is sub jec t has been w ritte n b y Shah and

Nathans on (1976). The v iru s has been iso la ted fro m the tissues and flu id s o f

pa tien ts w ith a v a r ie ty o f tu m o u rs . Two cases of PM L have yie lded v iru s e s

w h ich a re a n tig e n ic a lly in d is ting u ish ab le fro m SV40 bu t d is p la y m in o r DNA

d iffe re n ces confined to the h y p e rva ria b le reg ions of SV40 D NA (rev iew by

W e ine r and N arayan, 1974). SV40 was iso la ted fro m th re e m e ta s ta tic

m elanom as of a p a tie n t w ith no h is to ry o f va cc ina tion l ik e ly to con ta in SV40.

p le u ra l exudate contained SV40 a n ti-V and a n ti T an tibod ies (Soriano et a l.

1974). SV40 re la te d antigens, T and V w ere found in t lire e o f seven human

m en ing iom as when exam in ing the n u c le i of cu ltu red c e lls d e rive d f ro m the itf

Fus ion of these ce lls w ith SV40 p e rm is s iv e ce lls led to the appearance of

SV40 V antigen in a low percentage o f ce lls (Weisset a l . , 1975). I t has

a lre a d y been stated tha t the im m u n o lo g ica l re la tedness of SV40, BKV, JCV

and R FV , and the ex is tence o f s e ve ra l sub-tyiês w ith in each o f these, rende rs

the ta sk of d is tin g u ish in g these v iru s e s in iso la tion s o f human tum ou rs

re p o rte d p r io r to 1973 d if f ic u l t o r im p o ss ib le .

1 .2 .3 BKV, RFV and M M V

Since the v iru se s R FV and M M V are c lo s e ly re la te d to BKV, they w i l l

be d iscussed toge the r w ith BKV in th is section - M M V, RFV and BKV w i l l

c o lle c t iv e ly be ca lled BKV (H ow iey et a l. , 1975; M iao and D ougherty, 1977a)

and b)).

A su bs tan tia l num ber of pa tien ts subjected to long te rm im m u n o

suppress ion show evidence o f a c tive BKV in fe c tio n . G a rdne r e t a l. , (1971)

made the o r ig in a l is o la tio n o f BKV fro m the u rin e o f such a p a tie n t. T ha t

im m u n o lo g ic a lly c rip p le d pa tien ts , e ith e r due to the ra p y (k idney tra n s p la n t

p a tie n ts ) o r due to d isease syndrom es (e .g . W is c o tt-A ld r ic h syndrom e) show

r is in g antibody t i t r e s to BKV and u r in a ry e xc re tio n of the v iru s m ay be a

re s u lt o f p r im a ry in fe c tio n o r delayed la te n t in fec tio n (re v ie w by Padgett and

W a lke r, 1976). A n tibod ies to BKV a re p re sen t in 83% o f the human popu la tion

of England (G ardner, 1973) and a s im i la r fre qu e ncy has been found in the

U nited States (M u lla rk e y and Takem ato, 1973). The presence o f BKV in the

. 27.

human popu la tion is w idespread but the question of w hether BKV produces

d isease in man has not ye t been fu l ly answ ered. BKV DNA sequences were

detected in five o f tw e lve human tum ou rs and th re e o f fo u r human tu m o u r c e ll

lin e s (F io r i and d i M ayorca , 1976). In one k idney tra n sp la n t pa tien t, the

donor u re te r shov/ed f ib ro s is and an u lce ra te d e p ith e liu m , h is to lo g ic a lly

show ing la rg e in tra n u c le a r in c lu s io n s w h ich contained papovav irus p a rtic le s

when exam ined under the EM . (Colem an e t a l. , 1973; G a m e r e t a l . , 1971).

S im ila r obse iw ations have been made by ZuRhein and V a ra k is (1974). A r is ef

in an tibody t i t r e to BKV has been dem onstra ted in pa tien ts w ith G u il la in -B a rre

syndrom e (acute in f la m m a to ry po ly rad icL iloneuropa thy) in the absence of

s e ro con ve rs io n aga inst o the r candidate v iru s e s (Noordaa and W e ith e im -v a n

D ille n , 1977).

Tab le 4 l i s t the o ncogen ic ity o f BKV v iru s e s in an im a ls o th e r than man.

TA B LE 4: O N C O G EN IC ITY OF BKV VIRUSES IN N O N -H U M A N SPECIES

BKV Species I T u m o u r induced R eferences

BKV new born h am ste r sa rcom a Shah e t a l . , 1975

B KV (M M V )new born h a m s te r new born h am s te r

no tu m o u r no tum o u r

Takem oto e t a l , , 1974

BKV newborn h a m s te r fib ro s a rc o m a N oordaa 1976

B KV(M M V) new born h a m s te r ependym om a Costa et a l . , 1976

BKV(R FV) newborn h a m s te r sa rcom a D oughe rty 1976

BKV new born h a m s te r ep end ym om a and insu lin om a

U chida et a l . , 1976

The above su m m a ry ind ica tes tha t the BKV, MKIV and RFV group of

papovav iruses have a reduced p a th og e n ic ity and oncogen ic ity in the n a tu ra l

host and e xp e rim e n ta l a n im a ls when com pared w ith SV40 v iru s . T h e re m ay

be some d iffe re n ce s between BKV, M M V and RFV in th e ir o ncogen ic ity in

h a m s te rs .

2% ,

1 .2 .4 JC V iru s

U n like the BKV, M M V, RFV group o f v iru s e s above, th e re is s tro ng

evidence tha t JCV does cause d isease in man, its ' n a tu ra l host. JCV is

s tro n g ly associa ted w ith the ra re , dem ye lina tin g d isease, p ro g re ss ive

m u lt ifo c a l leukoencephalopathy (PM L). JCV p a r tic le s have been repea ted ly

dem onstra ted in the n u c le i o f o lig od e ifro cy te s in and around the le s io n both

b y E M and i m m un of lu o r e s cent s ta in ing ( fo r re v ie w , see Padgett and W a lke r,

1976). La te PM L les ions fre q u e n tly conta in g ian t a s tro cy te s m o rp h o lo g ic a lly

re s e m b lin g the m a lig n an t a s tro cy te s o f p le om o rp h ic g lio b la s to m a s (A s trom

et a l . , 1958; Z u rh e in , 1969). PM L is a disease com m on ly assoc ia ted w ith

pa tien ts show ing an im p a ire d im m une system e ith e r due to c h ro n ic d isease

o r im m unosupp ress ive the ra p y fo llo w in g k idney tra n s p la n ta tio n (Z u rh e in and

V a ra k is , 1974a; L e g ra in et a l . , 1974; Manz et a l . , 1971; Padgett and W a lke r,

1976). JCV has been iso la ted in a case of PM L w h ich a lso showed m u lt ip le

g lio m a s , s im i la r to the le s io ns produced in the h a m s te r fo llo w in g in tra c e re b ra l

ino cu la tio n of JCV (Castaigne e t a l. , 1974; W a lke r e t a l . , 1973). S eroep idem io logh

evidence ind ica tes tha t BKV and JCV c irc u la te co m p le te ly independently, su ppo rting

the v ie w tha t they a rc cgiite d is t in c t v iru s e s (Brown et al . , 1975).

JCV is oncogenic in an im a ls o th e r than man and fa b le 5 sum m a rise s

the in fo rm a tio n a va ilab le to date.

TA B LE 5: O N C O G EN IC ITY OF JCV IN N O N -H U M AN SPECIES

JCV s tra in Species T u m o u r induced R eferences

JC V (M A D i) new born ha m ste r m a lig n an t g lio m a * r a r e ly ependym om a

'W a lke r e t a l , , 1973

JCV(M AD2) new born h am s te r as above

JCV(M AD4) new born h am s te r 50% p in eo cy tom a .50% m a lignan t g lio m a j r a r e ly ependymoma^ and e x tra c ra n ia l n e u ro blastom a^ u n c la ss ifie d tum ou rs o f the ile u m

Padgett e t a l , , 1977

JCV new born h am ste r sa rcom as, m a in ly in te rn a l bu t some subcutaneous

Padgett and W a lke r, 1976,

-*

* inc lude m edu llob las tom a, g lio b la s to m a , u n c la ss ifie d p r im it iv e tum ou rs .

,2 9 .

W hile BKV and JCV v iru s e s are re ce n t d isco ve rie s , i t is a lre a d y c le a r

tha t each has se ve ra l v a r ia n ts o r s tra in s , each show ing d if fe r in g oncogen ic ity

in v iv o . I t is p robab le tha t the in fo rm a tio n p resen ted here does not

re p re se n t the com plete host range o r oncogen ic ity o f e ith e r v iru s but tha t

fu tu re w o rk w i l l expand p re sen t knowledge,

1. 2. 5 O ther V iru s e s of the Polyoma Group

(i) STMV

T lie s tum p ta ile d macaque v iru s (STMV) is a re c e n tly iso la ted papova

v iru s a lways p re se n t in the k idneys o f its n a tu ra l host, both in p o s t-n a ta l and

in foe ta l l i fe (Shah e t al , , 1977). I t is p resen t in un inocu la ted k idney c e ll

c u ltu re s o f the s tum p ta ile d macaque and has been shown to be d is ta n tly re la te d

to SV40 and BKV. It is unique among the papovaviruses in tha t m any v ir io n s

appear to possess an a dd itio n a l ou te r envelope (R e iss ig et a l. , 1976). No

in fo rm a tio n is yet ava ilab le in the li te ra tu re as to its in v iv o e ffec ts .

( i i ) Owl M onkey Papovavirus

One re p o r t (D an ie l e t a l. , 1976) describes the is o la t io n o f th is v iru s ,

tha t i t is not pathogenic fo r ra b b its and tha t a 100% m o r ta l i ty re s u lts w ith in

seven days fo llo w in g ino cu la tio n of newborn ham ste rs but w ith o u t s p e c ific

h is top a th o lo g ic le s io n s .

( i i i ) HD v iru s

HD v iru s was iso la ted fro m a perm anent m onkey k id n ey c e ll lin e

d e rive d fro m C ercop ithecuâe th iops m onkey (V E R O -ce lls ), I t has been shown

to have a s m a lle r d ia m e te r than SV40 under the EM , and i t s ' DN A has a

m o le c u la r w e igh t of 3.1 x 10^ daltons (com pared to 3. 6 x lO & fo r SV40 DNA^

(W aldeck and Sauer, 1977). No in fo rm a tio n is ye t ava ilab le as to its in v iv o

e ffec ts .

( iv ) Non Oncogenic P o lyom avirus es

E xc lud ing the STM V, HDV and the ow l m onkey papovav irus because of

th e ir re ce n t d isc o v e ry , a num be r o f the polyom a group of v iru s e s show no

oncogen ic ity .

, 3 0 .

K -v iru s is co m m on ly found in m ice and in new born m ic e ji t p roduces

pneum on itis and hepatic c e ll changes but not in adu lt m ice (F is h e r and K illia m ,

1953; K ilh am and M urph , 1953),

P orc ine Papovavirim hs not been shown to be oncogenic (Newman and Sm ith,

1972).

L a te n t Rat V iru s , o r the Toolan PI-1, H3 e tc. v iru s is now tliough t to be a

p a rv o v iru s bu t re m a in s a poss ib le papovavirus s ince 66% o f its DN A is double

s tranded. The v iru s has been found in a num ber of human tu m o u rs and is

o s te o ly tic ,s o ca lle d because i t causes M ongo lism in n eo n a ta lly inocula ted

ham ste rs (Toolan, 1960).

RKV V iru s

The ra b b it k idney va cuo la ting v iru s has been found in n o rm a l tissu es

as w e ll as Shope p a p illo m a s . I t is not known to be pathogenic in any species

(H a rtle y and Rowe, 1964; Ito e t a l. , 1968).

1.3 P a p illo m a v iru se s in v iv o - host range and oncogen ic ity

The p a p illo m a v iru s g roup o f the papovav iridae d is p la y a w ide range o f

oncogen ic ity in v iv o . Some m em bers produce benign e p ith e lia l h y p e rp la s tic

le s ions re s tr ic te d to th e ir host species w h ile o thers a re oncogenic in a num ber

of a n im a l spec ies. In gene ra l, those oncogenic m em bers produce tum ours

re s tr ic te d to e p ith e lia l and m esenchym al tissues and th e ir equ iva lents in the

c e n tra l nervous sys tem . It is now accepted tha t any one species m ay have a

num ber o f d if fe re n t and often p a r t ly re la te d p a p illo m a v iru s e s causing va rio u s

le s io n s . The fo llo w in g re v ie w w i l l con fo rm to the accepted p ra c tic e of

nam ing the v iru s e s acco rd in g to th e ir species of o r ig in a l iso la tio n .

1. 3. 1 Human P ap illom a V iru s (HPV)

p re v io u s ly , a ll pap illom as of man w ere thought to be due to one v iru s

(Andrewes and P e re ira , 1972). Recent l i te ra tu re now ind ica tes tha t th e re

m ay be s e ve ra l v iru s e s invo lved , some causing m o rp h o lo g ic a lly separable

le s io ns . Some le s io ns proceed to ca rc inom as and o th e r m a lignanc ies w h ile

o the rs re m a in benign. HPV has been shown to produce p ap illom as in the dog

(see Section 1. 3. 4(c) but in s u ff ic ie n t w o rk has been done to fu l ly d e te rm ine ■

. 3 1 .

the e xp e rim e n ta l host range of HPV, The on ly o the r re p o rts date fro m the

1930’s and show HPV to be n on -tra n sm iss ib le to dogs, guinea p igs , monkeys

and ca lves (B ibe rs te in , 1930).

1. 3. 2 V e rru c a v u lg a r is , p lana, p la n ta ris

T lie com m on cutaneous o r sk in p ap illom a can occu r on d iffe re n t

ana tom ica l s ite s , g iv in g r is e to d iffe re n ces in m a c ro sco p ic and m ic ro sco p ic

appearance and co rre sp on d ing p a tho log ica l te rm s (Review by Rows on and

Mahy, 1967). Papillom as a re com m on among school c h ild re n and young adults.

Tab le 6 i l lu s tra te s the e p id e m io lo g ica l nature of verrucas in Scotland fro m

1970 to 1976 (source Anon. 1977).

TA B LE 6: PREVALEN C E % OF VER R U C A IN SCOTTISH SCHOOL ENTRANTSAND SCHOOL LEAVER S FROM 1970 TO 1976.

YEAR SCHOOL ENTRANTS SCHOOL LEAVER S

Boys G ir ls Boys G ir ls

P* NT P N P N P N

1970 2. 39 44,9 2 .44 42.8 5. 67 32 .3 6. 24 32.4

1972 2.58 40.9 2 .37 39.2 5.87 33.8 6 .54 33.7

1973 2 .36 41,8 2.32 39.7 6. 10 32. 1 6 .37 32.1

1974 2.45 40.9 2. 52 39.5 5, 66 34 .6 5.91 34.5

1975 2. 12 37,7 2.43 35.5 9.89 19.2 8. 69 18.9

1976 2.20 37. 1 2. 27 35 .0 8.83 19.0 7.89 18.9

* P = p reva lence (%)

4- N = num ber exam ined (x lO ^)

Source - Anon. 1977.

Table 6 shows a p reva lence o f between 2, 12 and 2, 58% o f ve rrucc® in c h ild re n

of age 5 - 6 years on school e n try and between 5. 67 and 9. 89% o f v e r ru e aein

ch ild re n le a v ing school at 14 - 15 years o f age. There is a cons is tency in

these f ig u re s tha t suggests a stab le e p id e m io lo g ica l s itu a tio n w ith no

32

s ig n if ic a n t sex d iffe re n c e s . The p reva lence o f ve rrucas in the o ld e r age

g roups suggests tha t m ost, i f not a l l school le a ve rs w i l l have been exposed to

HPV by the t im e of p ub e rty . The fig u re s are in g ene ra l agreem ent w ith the

p reva lence of 7 - 10% re p o ite d in school c h ild re n by Rows on and Mahy (1967).

W idespread c o n tro v e rs y s t i l l e x is ts ove r the p re c is e m echanism of

re je c tio n o f v e rru c a and hence the value of v a rio u s im m u n o lo g ic a l pa ram ete rs in,

e s tim a tin g p re v io u s human exposure to HPV. D iffe re n t HPV antigen

p re p a ra tio n s have led to w id e ly d if fe r in g estim a tes o f a n t iv ira l an tibody in

pa tien ts w ith v e rru c a . A n t i HPV antibody was detected in 10 - 50% of

pa tien ts with, v e rru c a by O g ilv ie (1976) and 20 - 100% of pa tien ts w ith re g re ss in g

v e rru c a b y P yr honen (1976). Pyr: honen (1976) concluded tha t n e a r ly everybody

had been in fec ted w ith HPV by the age of 20.

In a com parison of the n o rm a l sk in su rface of p a ire d w a rt a ffected

and c o n tro l p a tie n ts , G lo o r e t a l. , (1976) found c e rta in s ig n if ic a n t d iffe re n ce s

(1) W a rt pa tien ts had a s ig n if ic a n t re d uc tio n in the percen tage amount of

squalene in the sk in su rface lip id s (2) W a rt pa tien ts had a reduced a lk a li

n e u tra lis a tio n t im e (3) The degree of w a te r m o is te n in g of the sk in was le ss in

w a rt pa tien ts .

In one th ird of p a tien ts , v e rru c a m ay p e rs is t fo r 7 -9 years (Culaie

and Bunney, 1976) w h ile the balance re g re s s w ith in tw o years (R u lison , 1942;

M assing and E ps te in , 1963). H ow ever v e rru c a m ay o ccu r fo llo w in g w a rt

re g re s s io n in up to one h a lf o f p a tie n ts . P ap illom as have o cca s io n a lly been

re p o rte d to cause e ro s io n of su rro u n d in g tissu e s , even bone (Shah et a l. ,1976),

how ever, m a lignan t tra n s fo rm a tio n o f sk in p ap illom as has neve r been

re p o rte d (Rowson and Mahy, 1967). T lie inc idence o f v e rru c a is increased in

pa tien ts w ith decreased c e llu la r im m u n ity as fo r exam ple , those who have

had re n a l tra n sp la n ts o r lym phom as (P e rry and H arm an, 1974; Spencer and

A ndersen, 1970; Reid et a l. , 1976).

1 .3 .4 Condylomata. acum inata

C ondylom a acum ina tum , the anogenita l w a rt o f man, m ay a ffe c t both

e p ith e lia l and m ucosal su rfaces and is com m on ly v e n e re a lly tra n s m itte d

(O rie l, 1971; L e v e r and Shaum b u rg -L e v e r , 1975). C ondylom ata are

s tim u la ted to g ro w and invo lve adjacent su rfaces d u rin g p regnancy and re g re s s

. 33 .

fo llo w in g d e liv e ry . Prevalence at d e liv e ry lias been es tim a ted at 1 - 2 %

(Cook et a l . , 1973). On occasions condylom a m ay become extensive g iv in g

r is e to the ra re g ian t condylom a of Buschke and Low enste in (1932), M a lignan t

tra n s fo rm a tio n o f condylom a has been re p o rte d re g u la r ly in the l ite ra tu re .

A nogen ita l w a rts a re associa ted w ith up to 5% of v u lv a l ca rc inom as and 15%

of pen ile ca rc in om as (Underwood and H ester, 1971; R hatigan at a l . , 1972),

O ccasional re p o r ts a lso show conve rs ion to m a lignancy of c e rv ic a l and

a no rec ta l condylom a (Kazai and Long, 1958; Sawyers, 1972; F itz g e ra ld and

H a m it, 1974). R a re ly , condylom a can invo lve the u re th ra (G artm an, 1956;

L in d n e r and P asq ie r, 1954; D re t ie r and K le in , 1975) and the b la dd e r

(Pettersson et a l. , 1976; K le im a n and Lan ca s te r, 1962; Lew is e t al . , 1962),

A nogen ita l w a rts have shown an in c re as ing p reva lence in re ce n t

years , and Ghosh (1977) c ite s a p reva lence of 5.34% pa tien ts p re se n tin g at

se xua lly tra n s m itte d d isease c lin ic s in 1976,

1 .3 .4 A lim e n ta ry and R e s p ira to ry T ra c t P ap illom a tos is

P ap illom as of the upper a lim e n ta ry and re s p ira to ry t ra c t fo rm a

com p lex of c lin ic a l and p a tho log ica l syndrom es often re fe r re d to using w id e ly

v a ry in g nam es. T hey a re grouped here fo r re v ie w purposes.

a ) F oca l E p ith e lia l H ype rp las ia

F o ca l e p ith e lia l h ype rp la s ia (FEH ) has been re p o rte d in the o ra l

c a v ity in man and chim panzee (see section 1. 1 .2) and a s im i la r le s io n occurs

on the bovine tea t (M eischke , 1978 and e lsew here in th is th e s is ). H is to lo g ic a lly ,

these les ions a re not p a p illo m a s bu t d is c re te papules o f h y p e rp la s tic e p ith e liu m

w h ich m ay coalesce to cover la rg e areas (P ra e to rius -C la use n , 1972).

P ap illom a v iru s has been iso la ted fro m a ll th ree an im a ls in w h ich the le s io n

has been re p o rte d (section 1. 1.2; Hanks et a l. , 1972; K u ffe r and P e ro l,

1976; P rae to rius-C lausen, and W ill is , 1971 and e lsew here in th is th e s is ).

The p reva lence of human FEH v a r ie s w id e ly in d if fe re n t p a rts o f the

w o r ld . South A m e ric a n I id ia n ch ild re n showed a p reva lence o f 33. 8%

(Soneira and Fonseca, 1964) and a re s tr ic te d popu la tion of G reen land ic

E sk im os of a ll ages, 35. 8% (P rae to rius -C lausen , 1970). A fa r lo w e r

p reva lence has been re p o rte d in Caucasians.. A x e l l / 1 9 7 found 0,11% o v e ra ll'

p reva lence in a la rg e su rve y of Swedes w ith the h ighest p reva lence of 0, 40% in

34.

people o ld e r than 75 years of age. In h is re v ie w , P ra e to riu s -C la u se n (1973)

concludes tha t the ae tio lo g y of FE H is v i r a l w ith a p ro b a b ility tha t a genetic

fa c to r a ffe c tin g s u s c e p tib ility to FE H is a lso invo lved.

b ) A lim e n ta ry T ra c t P ap illom a tos is

R eports o f o ra l condylom a acum inatum a re ra re and a re g e n e ra lly

associa ted w ith in fe c tio n fro m anogen ita l condylom as (Doyle et a l. , 1968;

Knapp and V ohara , 1967; S e ibe rt et a l. , 1969). O ra l m a n ifes ta tion s of

v e rru c a plana a re l im ite d to ep ide rm odysp las ia v e r ru c i fo rm is w h ich w il l be

d iscussed la te r .

O ra l v e rru c a v u lg a r is is a ra re le s io n among the gene ra l popu la tion

w ith a p reva lence of 0.10% re p o rte d by A x e ll (1976). H ow ever i t shows an

increased p reva lence in o ld people - a 6. 2% p reva lence was re p o rte d b y

Bhaskar (1968). M u lt ip le o ra l p a p illo m a to s is of p oss ib le gene tic ae tio logy is

seen in "nevus unius la te r is " and the " fo c a l d e rm a l h y p e rp la s ia sâidrorne"

(Brown and G o r lin , 1960; W ald on, 1970). M u ltip le p a p illo m a to s is o f o the r

p a rts o f the a lim e n ta ry t ra c t has a lso been re p o rte d , fo r exam ple the pa la te

(Lam bert e t a l. , 1976) and the oesophagus (D aran i and V il la , 1976). O nly one

re p o r t d esc rib e s the tra n s fo rm a tio n to m a lignancy of o ra l p ap illom as (Sam itz

et a l. , 1967).

c ) R e s p ira to ry T r a c t Pap i l l omato s is

P ap illom a to s is o f the la ry n x is the com m onest tu m o u r o f the la ry n x in

ch ild re n as w e ll as a s ig n if ic a n t d isease in adu lts (Bone et a l . , 1976). I t is a

p a r t ic u la r p rob lem because o f 1) the o b s tru c tive lo ca tio n 2) the m u lt ip l ic i ty

3) the hig'Ji re c u rre n c e ra te o f le s io n s and 4) the spread o f le s ions to the trachea

and b ro n c h i (A i - Saleem et a l . , 1968).

S evera l poss ib le modes of in fe c tio n have been pub lished . R eports show

some assoc ia tions (1) 42-62% of pa tien ts w ith la ryn g e a l p a p illo m a to s is had

co n c u rre n t o r p r io r sk in w a rts (Kaufm an and Baloch, 1969; Hajek, 1956)

(2) Some authors have suggested an assoc ia tion between canine p a p illo m a to s is

and human la ryn g e a l p a p illo m a s (Uhlm an, 1923; Isch ikaw a, 1936; R e s le r and .

Snow, 1967). ( i n a su rve y o f 100 dogs, Pyrhdnen (1976) found s e ro lo g ic a l

evidence o f HPV in fec tio n in a lm o s t 25%, J (3) L a ryng e a l p a p illo m a to s is in

c h ild re n has often been re p o rte d in assoc ia tion w ith m o the rs show ing condylom a

35,

d u r in g p regnancy and p a r tu it io n (W allenborn , 1976; Cook et: a l . , 1973).

F u r th e r evidence fo r the assoc ia tion o f la ryn g e a l and anogen ita l papd lom a tos is

coin.es^com parison o f v iru s DNA (Z u r Hausen et a l. , 1974; Delap et a l. , 1976;

G issn iann et a l. , 1977), v i r a l p ro te in s (P fis te r e t a l. , 1977), and im m un o lo g ica l

techniques (A lmeida, et a l. , 1969).

M a lignan t tra n s fo rm a tio n of la ryn g e a l p a p illo m a s has been reported

fo llo w in g ra d ia tio n th e ra p y (Fechne r e t a l. , 1974) but a lso in i t s ' absence

(Shapiro e £ a l , , 1976; Z chnder and Lyons, 1975).

1. 3. 5 E p id e rm o d ysp la s ia V e r ru c i fo rm is (EV)

W hile m u lt ip le p a p illo m a to s is o f poss ib le gene tic a e tio lo g y has been

m entioned e a r l ie r w ith re sp ec t to two syndrom es (see sec tion 1. 3, 4 (b)), EV

is s u ff ic ie n t ly w e ll stud ied to be tre a te d sepa ra te ly . EV is a d e rm a to s is

where v e rru c a p lana papules appear u s u a lly by p ube rty and d issem ina te to

a g re a t extent in e a r ly adu.lt l i fe (Jablonska and M ilew sky , 1957). HPV has

been id e n tif ie d in such les ions b y e le c tro n m ic ro s c o p ic and v iro lo g ic a l m ethods

(Yabe and Sadakane, 1975). The v i r a l in fe c tio n has been in c r im in a te d in the

e a r ly deve lopm ent o f sk in m a lignanc ies which appear in 25% of cases even

though HPV o r HPV DN A has not been detected in m a lig n an t c e lls (Yabe and

Sadakane, 1975; Jablonska et a l . , 1972 and 1976).

In ano ther genetic d isease, xe rode rm a p igm entosum , patien ts show

sk in cancers at an e a r ly age on lig h t exposed a reas due to de fec tive re p a ir

o f UV damaged DNA (C lea ve r, 1968; Robbins e t a l. , 1974). In EV pa tien ts ,

skm cancers a lso appear f i r s t on sun exposed areas and such pa tien ts a lso

show de fec tive re p a ir o f UV damaged DNA (H am m ar e£_al., 1976). HPV

e x trac te d fro m EV pa tien ts has been shown to be d if fe re n t f ro m HPV e x trac te d

fro m ve rru ca v u lg a r is le s io ns (O rth e t a l. , 1977). The a va ilab le evidence

supports the th e o ry tha t EV is a m u lt ifa c to r ia l d isease in w h ich HPV and

d e fec tive DNA re p a ir p la y im p o rta n t ro le s in gene ra ting m a lignanc ies .

P a ra lle l m odels e x is t in rabb.it Shope p a p illo m a to s is and bovine a lim e n ta ry

p a p illo m a to s is to be d iscussed la te r .

.36.

1. 3, 6 Shope Papilloma V irus

Shope p ap illom as a re com m on ly encountered in n i ld co tto n ta il ra b b its

(S y lv ilagus spp„ ), and e x p e rim e n ta lly , the v iru s produces p ap illom as in hares

(Lepus spp. ) and dom estic ra b b its (O ryc to lagus cuniculus)(Shope, 1933),

D om estic ra b b it p a p illo m a s a re poor p roduce rs o f v iru s , and fre q u e n tly

undergo m a lig n an t tra n s fo rm a tio n in to ca rc inom as (Rous and Beard, 1935).

T hey a re o f in te re s t as an e xp e rim e n ta l m odel, but a re not a n a tu ra lly

o c c u rr in g tu m o u r in the dom estic ra b b it (Shope 1933), A s such a m odel,

Shope p ap illom a v iru s in v iv o dem onstra tes a m a lig n an t p o te n tia l under the

r ig h t genetic and e nv iron m e n ta l in fluences. The presence o f p ap illom as is

re s tr ic te d to ra b b its in sta tes b o rd e rin g the M is s is s ip p i r iv e r even tliough the

suscep tib le host is spread w o rld w ide (F ried e w a ld and K idd, 1944),

A p p lic a tio n of ca rc in og e n ic chem ica ls (m ethy lcho lan th rene , ta r , p o ly c y c lic

hyd roca rbons) at o r p r io r to in fec tio n w ith v iru s causes a m o re ra p id

m a lignan t tra n s fo rm a tio n of p a p illo m a s in the dom estic ra b b it (Rogers and

Rous, 1951; Rous and F rie d e w a ld , 1944).

Shope p a p illo m a v iru s shows some genetic h e te ro g e n o ity dem onstra ted

by s e r ia l ly passaging s tra in s in dom estic ra b b its (Shope, 1935). Rous (1943)

suggested the presence of Shope v iru s v a ria n ts o r "ca n ce r p roduc ing v iru s e s "

caused the m a lig n an t tra n s fo rm a tio n o f p ap illom as but th is has not been

supported b y m o re re ce n t w o rk (Rogers et a l. , 1960).

1. 3. 7 R abbit O ra l P ap illom a V iru s (ROPV)

A lthough suscep tib le to ex iê rim en ta l in fe c tio n , w ild ra b b its have not

been re p o rte d to show o ra l p a p illo m a to s is w h ich o ccu rs in dom estic ra b b its .

The Shope v iru s (1. 3, 6) cannot induce p ap illom as in the o ra l c a v ity w h ile the

ROPV is s t r ic t ly l im ite d to the o ra l e p ith e liu m , M a c ro s c o p ic a lly and

m ic ro s c o p ic a lly , o ra l p a p illo m a s a re s im i la r to and o ccu r on the same s ite s

as foca l e p ith e lia l h yp e rp la s ia in man (section 1. 3. 4 (a)) (R o lzok e t a l. ,1966;

Parsons and K idd , 1943), In fe c tio n appears to depend on a n im a l contact

e s p e c ia lly f ro m doe to su ck lin gs . V iru s has been re co ve red fro m m ou th -

washings o f ra b b its fre e fro m p ap illom as (Andrew es and P e re ira , 1972),

No m a lignan t tra n s fo rm a tio n has been re p o rte d .

. 37 .

1. 3. 8 Canine Papilloma V irus

A p a p illo m a v iru s has been id e n tifie d in one case of cutaneous

p a p illo m a to s is in the dog (W atrach, 1969) and v i r a l inc lus ions have been seen

in the s u p e r f ic ia l e p ith e lia l la y e rs (A lliso n , 1965),

1. 3, 9 Canine O ra l P ap illom a V iru s

O ra l p a p illo m a to s is is p r im a r i ly a d isease of young dogs and appears

to be lim ite d to the mucous m em branes of the eye and o ra l c a v ity , though i t

m ay a ffe c t sk in im m e d ia te ly adjacent to mucocutaneous junc tions of these

areas (C ham bers and Evans, 1959; C ham bers et a l. , 1960). One case has

been re p o rte d o f m a lignan t tra n s fo rm a tio n (W atrach et a l. , 1970) „ O ther

species appear to be insuscep tib le (Cham bers and Evans, 1959 and re fe ren ce s

th e re in ). The v iru s o f canine o ra l p a p illo m a to s is is thought to be d is t in c t

f ro m tha t o f sk in p a p illo m a to s is (A llis o n , 1965).

E 3.10 Equine P ap illom a V iru s

Skin papillom as on horses have long been recogn ised . The v iru s has

been iso la ted and d ie d isease tra n s m itte d to o the r ho rses . The tum ours

m a in ly invo lve the nose and lip s o f young horses but m ay a ffe c t o th e r p a rts o f

the in tegum ent. T lie v iru s does not a ffec t ca lves, lam bs, dogs, ra b b its o r

guinea p igs . (Cook and O lson, 1951; B rune r and G ille s p ie , 1973). Equine

sa rco id w i l l be d iscussed w ith Bovine Papilloma. V iru s (BPV) la te r .

1 .3 . 11 Goat P ap illom a V iru s

Tw o re p o rts descrü ie p a p illo m a to s is in goats. The f i r s t by D av is

and K em per (1936) re p o rts ben ign w a rts on a n te rio v e n tra i sk in s ite s . The

o th e r desc ribes w a rts on the udders o f goats, some shoiving m a lignan t

tra n s fo rm a tio n to ca rc inom as (M oulton, 1954).

1 .3 .1 2 H a m ste r Papi Horn a V iru s (Graf.fi)

A v iru s s im i la r in s ize and DNA content to p o lyom a v irus was found

in the k e ra tin ize d c e lls of h a m s te r p ap illom as of e p id e rm a l h a ir fo l l ic le

o r ig in (G raf.fi et a l. , 1970). On tra n s m is s io n to ham ste rs i t caused e p id e rm a l

p ap illom as , a 30 - 80% incidence of lym phom as and leukaem ias w ith in

36

4 - 8 weeks, and a few subcutaneous sa rcom as, k i ra ts , ino cu la tio n

produced re t ic u lu m c e ll sa rcom as w h ile in newborn ra b b its , f ib ro m a s

developed at the s ite o f in o cu la tio n . A co m p lica ting fa c to r has been the

is o la tio n of C -type v iru s p a r tic le s in h a m s te r lym phom as and sarcom as

induced by the G ra f f i v iru s (G ra ff i et a l. , 1963 and 1967),

I . 3. 13 Sheep P ap illom a V iru s

Benign sk in fib ro p a p illo m a s have been re p o rte d on the sk in o f sheep

(Head, 1965). A tte m p ts a t tra n s m is s io n of v iru s to sheep and c a tt le have been

unsuccessfu l but new born h am s te rs inocu la ted w ith the v iru s developed

fib ro s a rc o m a s (G ibbs, p e rson a l com m un ica tion , c ited b y B artho ld , 1974).

Sheep inocu la ted w ith bovine p a p illo m a v iru s d id not develop le s io ns (Barthold,

1974).

1.3. 14 D e e r F ib ro m a V iru s

Contagious cutaneous tum ou rs have been re p o rte d in the deer, e lk , and

moose (Fay, 1970), The tum ou rs in the deer are caused b y the dee r fib ro m a

v iru s and are p re d o m in a n tly fib ro m a to u s f ib ro p a p illo m a s (Shope et a l. , 1958).

T here is one re p o r t of m a lignan t tra n s fo rm a tio n w ith p u lm o n a ry m e tas tas is

(K o ile r and O lson, 1971). The v iru s is on ly p re sen t in the o u te r e p ith e lia l

p o rtio n s of the tu m o u r in dee r and causes fib ro s a rc o m a s on in ocu la tion in

h a m s te rs . No response fo llo w s in o cu la tio n of v iru s in to horses o r ca ttle

(K o ile r and O lson, 1972; T a jim a e t a l. , 1968).

1 .3 .1 5 Equine S arcoid

Equine sa rco id is a fib ro m a to u s o r a fib ro p a p illo m a to u s tu m o u r o f the

sk in o f horses w h ich has long been suspected o f be ing in fe c tio u s (Ragland e t a l. ;

1970). A p a p o va v iru s has been iso la ted but a v iru s un like the papovav iridae

has been re p o rte d fro m c e ll c u ltu re s de rived fro m sa rco id (A llis o n , 1965;

England et a l. , 1973). V a r ia b le re s u lts are re p o rte d f ro m tra n s m is s io n

expe rim en ts (Ragland et a l. , 1970). Bovine p a p illo m a v iru s (BPV) causes

s a rc o id - lik e tu m o u rs in the horse (Olson and Cook, 1951) and recen tly , BPV

s p e c ific DNA sequences have been detected in n a tu ra lly o c c u r r in g equine

tu m o u rs (L a n ca s te r c t a l . , 1977).

.39

1 .3 .1 6 Bovine Papilloina V irus (BPV)

Bovine pap illom a v iru s has been e x trac te d fro m a v a r ie ty o f les ions

o c c u rr in g on d if fe re n t a na tom ica l s ite s in c a ttle . BPV was found to be the

causative agent o f cutaneous fib ro p a p illo m a s by C reech (1929); gen ita l

f ib ro p a p illo m a s (M cEntee, 1952); and i t m ay be associated w ith ch ro n ic

enzootic h ae m a tu ria o f c a ttle (O lson et a l. , 1965). A ty p ic a l p ap illom as have

been shown to conta in BPV (Bartho ld e t a l. , 1974). BPV has been extracted

f ro m a lim e n ta ry p ap illom as (J a rre tt et a l . , 1978). T h is thes is w i l l re p o rt the

e x tra c tio n and tra n s m is s io n of BPV fro m th ree types o f te a t le s io n s - f ib ro -

p a p illo m a , p a p illo m a and fo ca l e p ith e lia l h ype rp la s ia (M e ischke , 1978).

E x p e rim e n ta lly , BPV induces m ening iom as in ca lves (G ordon and Olson,

1968); po lypo id tum ou rs of the u r in a ry b ladde r of c a ttle (O lson et a l. , 1959);

s a rc o id - lik e le s io ns in the horse (Olson and Cook, 1951); f ib ro b la s t ic tum ou rs

in the h a m s te r (B o iron et a l . , 1964; C h e v ille , 1966; L a sn e re t et a l. , 1965;

Robl e t a l . , 1972; Robl and O lson 1968), and f ib ro m a to u s tum ou rs in C3H/eB

m ice (Boiron et a l. , 1964).

1 .3 . 17 O ther Pap illom a V iru se s

Cutaneous p ap illom as have been rep o rte d on Cebus (Lucke et a l . , 1950)

and Rhesus (Sm ith et a l. , 1972) m onkeys. The fo rm e r was tra n s m is s ib le to

old and new w o rld m onkeys. V iru s has not been iso la ted , but pap illom as have

a lso been re p o rte d on Macaca (Brown e t a l. , 1972) and C o l obus (Boever and

K e rn , 1976) m onkeys.

P apovaviruses have been seen by EM fro m p a p illo m a s on V irg in ia

Opossum (K o ile r, 1972); ^ on the cha ffinch and re la te d species (O sterhaus

et a l. , 1977). A tra n s m is s ib le g en ita l p a p illo m a to s is occurs in p igs (P a rish ,

1961 and 1962). P ap illom as found on f is h appear to be associa ted w ith a v iru s

o th e r than the papova group (W e llings and C hu inard , 1964) w ith the poss ib le

exception o f the e p id e rm a l p ap illom as in Salmon (C h ronw a ll, 1976).

1 ,3 ,1 8 S um m ary - P apovaviruses in v ivo

Table 7 l is ts the papovav iruses, th e ir n a tu ra l hosts and th e ir

oncogen ic ity under n a tn ra l and e xp e rim e n ta l cond itions .

. 40 .

TA B LE 7: P apovaviruses in v iv o -- host range and oncogen ic ity

V iru s O ncogen ic ity in N a tu ra l Host E xp e rim e n ta l O ncogen ic ity

Polyom a c a rc in o m a s , adenom as, sa rcom as in mouse

ham s te r: sa rcom as and m en ing iom as; sarcom as and f ib ro m a s in ra ts , ra b b its , m astom ys, fe r re ts and guinea p igs

SV40 fib ro s a rc o m a s in mon.keys c e n tra l nervous system f ib ro b la s t ic neoplasm s in h am ste rs and m astom ys. lym phosa rcom a and re t ic u lu m c e ll sa rcom as in weaning h a m s te rs

BKV, RFV M M V

associa ted w ith f ib ro b la s t ic neoplasm s in the ce n tra l nervous sys tem of man. ra re ly , a ffec ts u ro g e n ita l e p ith e liu m

produce f ib ro b la s t ic neop lasm s of CNS and e ls e w here in new born h am s te r

JCV associa ted w ith PAIL and p o s s ib ly g lio b las to m a s in man

new born h a m s te r -d if fe re n t ia l onco g en ic ity between v iru s s tra in s , p ineocytom as and f ib ro b la s t ic neoplasm s of b ra in . Sarcom as in non CNS s ite s

K V iru s pneum on itis and l iv e r damage in new born m ice , no t oncogenic

no t oncogenic

Porc inePapovavirus

no t oncogenic in p igs not known

RKV not oncogenic in ra b b its not pathogenic o r oncogenic

HDVSTMV

not known not known

not known not known

. 4 1 .

TABLE 7: (Cont'd) Papovav iruses in vivo - Host Range and Oncogenicity

V iru s O ncogen ic ity in N a tu ra l Host E xp e rim e n ta l O ncogen ic ity

HPV F o ca l e p ith e lia l hype rp la s ia , ben ign p a p illo m a s and under c e rta in cond itions , ca rc inom as

M ay be tra n s m is s ib le to dogs, p ro du c in g g en ita l pap illom as

Shope p ap illom as and ca rc inom as under c e r ta in cond itions in w ild ra b b it .

p a p illo m a s and ca rc inom as in dom es tic ra b b it

ROPV benign p ap illom as in dom es tic ra b b it

not known

Canine Skin P. V .

benign p ap illom as in dog not known

Canine O ra l P.V.

o ra l p ap illom as in dog r a r e ly ca rc in om as

o the r species appear to be insu scep tib le

Goat Skin P .V .

cutaneous p ap illom as o cc a s io n a lly ca rc in om as in goat

not known

G ra ff i p a p illo m a s in ham ste rs new born h am s te rs - subcutaneous sa rcom as ; fib ro s a rc o m a s in ra b b its ; re t ic u lu m -c e ll sa rcom as in ra ts

Sheep P. V , f ib ro p a p illo m a s in sheep fib ro s a rc o m a s in h am ste rs

D eer F ib ro m a V iru s

cutaneous f ib ro m a s , f ib r o p ap illom as ra r e ly m a lignanc ies

fib ro s a rc o m a s in ham ste rs

BPV cutaneous p ap illo m a s , f ib r o p a p illo m a s a lim e n ta ry p a p illo m a s te a t p ap illom as f ib ro p a p illo m a s and foca l e p ith e lia l h ype rp la s ia al im entar y ca rc in om a s , adenocarc inom as under c e rta in cond itions

m en ing iom as in ca lves; u r in a ry b la dd e r pol)p)s in ca ttle ; s a rc o id - lik e le s io n s in ho rse ; f ib ro b la s t ic tum ou rs in h a m s te r and m ouse.

42.

1.4 Papovav iruses In V itro

M ost m em bers o f the papovav iridae have the a b i l i ty to tra n s fo rm c e lls

in v i t r o . A num ber w i l l do so w it li ju s t th e ir DNA - in some cases th is is

m ore e ff ic ie n t s ince the caps id p ro te in s m ay h in d e r DNA e n try in to the c e ll,

w h ile in o the r cases, DN A induced tra n s fo rm a tio n is fa r less e ff ic ie n t than

whole v iru s tra n s fo rm a tio n .

When papovav iruses in fe c t c e lls , in v ivo o r in v i t r o , two modes m ay

re s u lt, v iru s p ro du c tio n o r c e llu la r tra n s fo rm a tio n . C e lls w h ich produce

v iru s a re ca lled p e rm is s iv e . C e lls w h ich tra n s fo rm do no t n o rm a lly re lease

v iru s and in some cases liave neve r been shown to re le ase v iru s . Tab le 8

l is ts the c h a ra c te r is t ic s o f tra n s fo rm e d c e lls . Some tra n s fo rm e d ce lls

d is p la y a ll the c h a ra c te r is t ic s lis te d but m ore com m on ly papovav irus

tra n s fo rm e d c e lls on ly d is p la y some of them .

TA B LE 8: PR IN CIPAL C H A R A C TE R ISTICS OF T RANSF ORMED C E LLS

(source Harnden, 1976)

1. V a r ia b i l i t y in m o rpho logy of in d iv id u a l ce lls

2. Tendency to g ro w in a ra n d o m ly o rie n te d m anner even when incontact w ith o th e r c e lls ,

3. Tendency to fo rm m u ltip le la y e rs o f c e lls .

4. Low re q u ire m e n t fo r se rum in the m edium

5. A b i l i t y to fo rm co lon ies in so ft agar

6. Ind e fin ite life s p a n in c u ltu re

7. Presence of new su rface a n d / o r nuc lea r antigens

8. A lte re d d is tr ib u t io n o f b ind ing s ites fo r p la n t le c tin s

9. Presence of ch rom osom e a b n o rm a lit ie s

10. A b i l i t y to produce tum ou rs on inocu la tion w ith a p p ro p ria te host.

1. 4. 1 Polyom a and SV40 In V it ro

P o lyom av irus w i l l tra n s fo rm mouse, ra t, ra b b it, guinea p ig , dog;

ca ttle , h a m s te r, m onkey and hum an c e lls in v i t r o . In fec tious v iru s cannot

n o rm a lly be re co ve re d fro m c e lls w ithou t the aid of agents such as ,U l t r a - '

V io le t (UV) lig h t o r m ito m y c in C o r the fus ion of tra n s fo rm e d c e lls to c e lls

p e rm is s iv e fo r re p lic a tio n (Black, 1968; Foge l and Sachs, 1969). Polyom a

. 43

v iru s n o rm a lly on ly re p lic a te s in cu ltu re s of mouse o r ig in and o ccas ion a lly in

r a t tnm ou r tissue c u ltu re s (Andrewes and P e re ira , 1972). In p e rm is s iv e

c e ll m ono laye rs , polyom a causes plaques due to ly t ic in fe c tio n . N a tu ra lly

o c c u rr in g plaque v a r ia n ts have been described causing d if fe re n t plaque types,

SV40 can tra n s fo rm p ig , ra b b it, mouse (B lack and Rowe, 1963), ra t

(D ide rho lm ec a l . , 1966), m onkey (Fernandes and M oorhead, 1965) and human

c e lls (Shein and E nders, 1962; K op ro w sk i et a l . , 1962) under in v i t r o conditions.

F o llo w in g exposure o f hum an c e lls to SV40 both tra n s fo rm a tio n and v iru s

re p lic a tio n occu r and so the ce lls a re ca lled se m lp e rm is s iv e fo r SV40,

SV40 re p lic a te s m ost e ff ic ie n t ly in m onkey c e ll c u ltu re s .

Both polyom a and SV40 have n a tu ra lly o c c u rr in g and e x p e r im e n ta lly

induced m utant s tra in s . T e m p e ra tu re sensi t ive m u tan ts w h ich a ffe c t m ost i f

not a ll genes re q u ire d fo r ly t ic in fe c tio n have been iso la ted . These m utants

a re se lected on the basis of e ffec ts in ly t ic in fec tio n at the re s tr ic t iv e

te m p e ra tu re . V iru s re p lic a tio n can be defective at a num ber o f stages,

de te rm ined by m easuring the induction of c e llu la r DNA syn thes is , v i r a l DNA

syn thes is and the appearance o f v i r a l antigens o r p a r tic le s .

Host range m utants are de fec tive in v ir a l functions re q u ire d fo r

tra n s fo rm a tio n . They w ere o r ig in a l ly se lected fo r th e ir a b i l i ty to g row on

tra n s fo rm e d mouse c e lls . So fa r m ore than a dozen host range m utants have

been shown to be unable to tra n s fo rm mouse, ra t o r h a m s te r c e lls .

Defe c t ive m utants have d e le tions , in s e rtio n s , subs titu tion s o r o the r

re a rra ng e m e n ts in th e ir DNA. G e n e ra lly they w i l l not re p lic a te w ithou t the

use o f a nondefective h e lp e r v iru s o r DNA.

A denov i r us-SV40 hy b r id v i r uses a re adenoviruses w h ich conta in

cova len t genome in s e rtio n s of SV40 DNA. They w ere o r ig in a l ly iso la ted

becauseâdenoviruses do not re p lic a te e ff ic ie n t ly in m onkey ce lls unless the

c e lls a re co in fected w ith SV40. The SV40 DNA in s e rtio n s e x e rt a he lpe r

function re n d e rin g the h yb rid v iru s non -de fec tive fo r re p lic a tio n in m onkey

c e lls . D iffe re n t antigens a re expressed in ce lls in fected by the va rio us

h yb rid s suggesting tha t two o r m ore p a rts o f the SV40 DNA m ay be invo lved ,

A num ber o f rev iew s on SV40 and polyom a in v i t r o and the genetics

o f these v iru s e s have been pub lished re c e n tly (E ckh a rt, 1977; F r ie d and

. 44 .

G r if f in , 1977; K hou ry and Salzm an, 1975; W illia m s , 1976; Rows on, 1976).

1. 4. 2 BKV (R FV , M M V) in v it r o

Tab le 9 l is ts the c e lls known to be p e rm is s iv e fo r BKV re p lic a tio n .

BKV produces cy topa th ic e ffec ts (CPE) but no p rogeny in p r im a ry Shesus

m onkey k idney c e lls (G a rdne r e t a l. , 1971), p r im a ry A fr ic a n green monkey

c e lls , and two c e ll lin e s d e rived fro m the la t te r (Takem oto and M u lla rke y ,

1973).

TABLE 9: C E LLS PERMISSIVE FOR BKV R EPLIC ATIO N

Source

Human

C e ll Type References

Monkey

p r im a ry fo e ta l k idney p r im a ry foe ta l f ib ro b la s t p r im a ry foe ta l l iv e r and lung- p r im a ry foe ta l b ra in (g lia l) W1 - 38 ce ll line foe ta l k idney foe ta l lung foe ta l b ra inVERO (A fr ic a n green monkey k idney)

D oughe rty and D i Steffano, 1974 P o rto la n i e t_ a l., 1975 Seehafer et a l. , 1975 L e ca rta s e t a l . , 1974; Takem oto and M u lla rkey^ 1973 D oughe rty and DiSteffano,1974 C olem an et a l. , 1973 Takem oto e t a l. , 1974 Takem oto and M u lla rke y , 1973

Table 10 l i s t the c e lls tha t have been shown to be tra n s fo rm a tio n

se ns itive to BKV in fec tio n .

TABLE 10: C E LLS T H A T ARE BKV TR AN SFO R M ATIO N SENSITIVE

Source

H am ste r

Rat Rabbit Nude Mouse

C e ll Type R eferences

p r im a ry foe ta l f ib ro b la s t p r im a ry foe ta l k idney BHK -21 ( lia m s te r k idney) b ra inp r im a ry k idney ce lls p r im a ry k idney k idney

D ougherty , 1976 P o rto la n i et a l. , 1975 M a jo r and D iM ayo rca 1973 Tanaka et a l . , 1976 V ander Noo:rda 1976 Mason and Takem oto , 1977 Costa et a l. , 1977

45.

Some BKV tra n s fo rm e d c e lls (e .g . h am s te r k idney) are tum < jrigen ic in v ivo

w h ile o tlie rs (e .g . ra b b it p r im a ry k idney) are not. D iffe re n t BKV re la ted

antigens are expressed in d if fe re n t tra n s fo rm e d c e lls and some authors

suggest tha t d if fe r in g BKV s tra in s (A/IlvIV, RFV) may v a ry b io lo g ic a lly

(Mason and Takem oto , 1977). T ra n s fo rm a tio n w ith BKV DNA o f ham ster

and ra t c e lls has been achieved w ith g re a te r frequency and s h o r te r tim e than

w ith in ta c t v iru s (van de r Noorda^ ,1976). T h is suggests tha t BKV v ir io n s do

not become un coated o r pene tra te e ff ic ie n t ly in roden t c e lls (Takem oto and

A/iartin, 1976).

1 .4 .3 JCV il l v i t ro

In co n tra s t to BKV, SV40 and po lyom a, JCV has an e x tre m e ly

re s tr ic te d c e ll range. JCV grow s and produces CPE o n ly in p r im a ry foe ta l

human g lia l (PHFG) c e ll c u ltu re s tha t conta in a h igh p ro p o rtio n o f spongioblasts,

I t m ay take 6 - 9 m onths post in fe c tio n fo r CPE to be observed in PHFG us ing

JCV fro m diseased b ra in . JCV is s tro n g ly c e ll associated and cu ltu res

re q u ire m echan ica l d is ru p tio n o r re c e p to r des troy ing enzym e (RDE) to fre e

v iru s fro m c e ll d e b r is . A f te r s e r ia l passage in c u ltu re , CPE appear 12 - 14

days post inocu la tion (Padgett and W a lke r, 1976). Padgett et a l . , (1977)

a ttem pted to in fe c t a w ide range o f c e ll types fro m hum an, p r im a te , ham ste r,

m in k and mouse o rig in s w ithou t success e ith e r in the fo rm of JCV re p lic a tio n ,

CPE o r tra n s fo rm a tio n .

1. 4„ 4 O ther P o lyom aviruses in v i t r o

a) The K -v iim s o f m ic £ has been shov/n to cause tra n s fo rm a tio n in

e m bryon ic mouse lung, 6 weeks post in fec tio n . One lin e of tra n s fo rm e d ce lls

has produced tum ours in new born o r ir ra d ia te d m ice (Takem oto and Fab isch ,

1970, c ited by A ndrew es and P e re ira , .1972),

b) R abbit K idney Vacuo la tin g V iru s (R KV)produces c e ll-v a c u o la tio n

in p r im a ry ra b b it k idney c e ll c u ltu re s , CPE fo rm on m ono laye rs but c e lls of

o the r species appear insuscep tib le (H a rtle y and Rowe, 1964), Mason and

Takem oto (1977), re p o r t g row th of RKV on BKV tra n s fo rm e d ra b b it k idney

cu ltu re s and used the technique to plaque t it ra te RKV.

46 ,

c) I I D v iru s lias been re p o rte d to tra n s fo rm the A fr ic a n green

m onkey k idney c e ll line ,V E R O . (W aldeck and Sauer, 1977),

d) The o w l m onkey papovav irim has been re p o rte d to in fe c t on ly

ow l m onkey k idney, cornea and i r i s c e ll cu ltu re s (D anie l e t a l . , 1976).

e) S T M V iru s has been rep o rte d to re p lic a te and produce CPE in

p r im a ry k idney cu ltu re s fro m the s tum p ta iled Macaque and Rhesus m onkeys,

STMV is a n tig e n ic a lly re la te d to SV40 and BKV but no re p o r ts o f in v i t r o

tra n s fo rm a tio n have been made. (R e iss ig e t a l. , 1976),

1. 4. 5 Human Papil lo m a V iru s (HPV) in v it r o

A num ber o f re p o rts c la im to have grow n HPV in tissu e c u ltu re .

Mendel son and K lig m a n (1961) inocula ted m onkey k idney c e ll c u ltu re s w h ich

a fte r the th ird passage, s ix weeks post inocu la tion , showed CPE. Inocu la tion

of u lt ra f i l t r a te s o f tissu e c u ltu re f lu id in to tw enty human sub jects and observed

t lie deve lopm ent o f v e rru c a a t the in o cu la tio n s ite in 50% o f sub jects 3 to 12

m onths post ino cu la tio n . Subsequent re p o rts by M adpherson (1962); M organ

and Balduzzn (1964); O rosz lan and R ich (1964); and Noyes (1965) have not been

co n firm e d (Butel, 1972). F u rth e rm o re , when v iru s con ta in ing ve rru ca e are

grow n in -v itro , evidence o f v i r a l re p lic a tio n by e le c tro n m ic ro sco p e and

im m u n o h is to lo g ica l methods is lo s t (N irm u ra cjâR , 1975),

The e a r l ie r obse rva tion o f B u te l (1972) tha t HPV in fec ted confluent

m ono laye rs o f human em b ryo n ic k idney c e lls show a s tim u la tio n of DNA

syn thes is , was supported by L a n ca s te r and M einke (1975) who re p o rte d HPV

DNA p e rs is ten ce in c u ltu re s of a hum an fo re s k in d e rive d c e ll lin e .

E is ii ig e r e t al . , (1975) re p o rte d HPV induced re p lic a tio n and CPE in

a, human e p ith e lia l c e ll lin e (B. E . ). They co n firm e d th is us ing im m u n o lo g ica l, ■

EM and ra d io - la b e ll in g techn iques. In ano ther re p o r t o f the same e xpe rim en ts ,

Hadden (1975) em phasised tha t HPV p ro du c tio n was lo w us ing BE ce lls and

tha t a search fo r m ore p roduc tive c e lls had com m enced. As w ith the e a r l ie r ,

re p o rts , these fin d ing s aw a it c o n firm a tio n .

O nly one re p o r t d e sc ribe s HPV induced tra n s fo rm a tio n o f p r im a ry

human foe ta l sk in and m uscle c u ltu re s (Noyes, 1965), but th is has not been

supported by o th e r au tho rs .

. 47 ,

1. 4, 6 Shope Papilloma V irus in v i t ro

Shope v iru s has n eve r been grown in v it ro . No changes w ere

re p o rte d in c e lls fro m ra b b it sk in to which v iru s had been adsorbed, but

these gave r is e to p ap illom a when tra n s fe rre d to h am ste r cheek pouches.

N u c le a r v iru s s p e c ific antigen d isappeared fro m p a p illo m a d e rive d ce lls

held a t but reappeared at 30^C in the cy top lasm (S h ira to r i e t a l , , 1969).

V a rio u s authors have s tud ied in v it r o cu ltiva te d c e lls fro m Shope

induced p ap illom as (Coman, 1946; Ish im o to et a l . , 1970; Ish im o to and Ito,

1971) which do d is p la y c e r ta in an tigen ic s im i la r it ie s seen in in v iv o le s io ns .

In v it r o in fe c tio n of ra b b it c e lls w ith Shope v iru s has a lso been stud ied and

i t has been shown by im m uno fluo rescence tha t v iru s does in fe c t bu t not

re p lic a te ^c e ils (De M aeyer, 1962; K re id e r e t a l . , 1967; M ou lton and Lau,

1964), Shope v iru s in fected c e lls do not show m o rp h o lo g ica l changes but the

v iru s is able to a lte r the p e rm is s iv ity of ra b b it c e lls to SV40 p en e tra tio n

(Chardonnet e t a l . , 19767V

1, 4, 7 Bovine P ap illonn i V iru s (BPV) in v i t r o

BPV has been found to tra n s fo rm bovine k idney and foe ta l bovine

con junc tiva (DBG) c e ll lin e s , secondary cu ltu re s o f fo e ta l bovine h e a rt

(B lack et a l . , 1963), foe ta l bovine sk in c e lls , p r im a ry c u ltu re s of foe ta l

bovine sk in p ieces, secondary f ib ro b la s t ic cu ltu res of v a rio u s s tra in s o f f o e t a l j.iOLise E rh o t ia s o t a l . , l g u ÿ i g 6 4 j amdfoe ta l h a m s te r c e ll c u ltu re s (G era ides, 1969). BPV DNA has a lso been shown

to tra n s fo rm foe ta l bovine sk in c e lls (Bo iron e t al . , 1965),

Olson e t^ a l. , (i960) c la im e d to have grown BPV in the c h o r io a lla n to ic

m em brane (CAM ) of fe r t i le ch icken eggs. These re s u lts aw a it c o n firm a tio n .

(See a lso Bagdonas and O lson, 1954). O lson et a l . , (1960) used BPV g row n

on ch icken C AM to vacc ina te ca lves but w ithou t success. T h is thes is w i l l

re p o r t tra n s fo rm a tio n of foe ta l bovine cu ltu re s o f va rio u s tissu es and a ttem pts

to c u ltiv a te BPV bi_vh:ro.

1 .4 .8 O ther P ap illom a V iru s e s in v it r o

T he re are no re p o r ts in the li te ra tu re o f re p lic a tio n , tra n s fo rm a tio n

o r CPE fo rm a tio n in v i t r o by any of the o th e r p ap illom a v iru s e s lis te d in '

. 48

section 1.3 o f th is th e s is . T h is is c e r ta in ly p a r t ly because, as w ith the

new er p o iyo m a v iru se s , in s u ff ic ie n t re s e a rc h has been com pleted. S trong

candidates fo r in v i t r o tra n s fo rm a tio n of host ce lls m ust inc lude the deer

fib ro m a v iru s and the sheep fib ro p a p illo rn a v iru s . L ike m any e a r l ie r

re v ie w e rs (Rowson and Mahy, 1967) th is author is tem pted to conclude tha t

no p a p illo m a v iru s has been cu ltiva te d in v i t r o . H ow ever, the re p o rts of

successfu l HPV o r BPV (the two m ost studied p a p illo m a v iru s e s ) re p lic a tio n

in v i t r o are m any, are spread over 15 years and come fro m w id e ly va rie d

sources. The p r o b a b le conc lus ion is tha t HPV and BPV have indeed been

cu ltiva te d in v it r o , but in e ff ic ie n t ly and under exceptiona l cond itions .

1. 5 _Im tm inolog ic a l A spects o f Papovavirus In fection

Papovaviruses them se lves a re composed o f m a jo r capsid p ro te in

(VPl); two m in o r s tru c tu ra l p ro te in s (VP2 and VP3), at le a s t fo u r d if fe re n t

host d e rived h i stones w h ich w rap the v ir a l genome and the supe rco iled DNA

of a p p ro x im a te ly 5, 000 base p a irs . C e ll in fec tio n by v iru s is accom panied

by changes on the c e ll su rface and w ith in the c e ll cy top lasm , nuc lea r m em brane

and nucleus. When v iru s o r tra n s fo rm e d ce lls are inocu la ted in to a pp rop ria te

hosts, they m ay im m un ise the host aga inst fu r th e r inocuk itions . E x a c tly

which antigens a re expressed by v iru s in fected c e lls depend la rg e ly on the

degree to w h ich in fec tio n has p rog ressed to the endpoints o f re p lic a tio n o r

tra n s fo rm a tio n , but a lso on the host response to these changes. T liose

v iru s e s m ost e a s ily cu ltiva te d in v it r o a re a lso those about w h ich the m ost

im m un o lo g ica l in fo rm a tio n is ava ilab le . T h is re v ie w w i l l on ly b r ie f ly

ou tline the m a jo r fea tu re s o f the p o lyo m a v iru s group re la te d antigens befo re

pu rsu in g the p a p illo m a v iru s e s in g re a te r d e ta il.

1 .5 .1 P o iyom aviruses - Im m unol og ica l Aspects

It is poss ib le to produce sp ec ific a n tise ra aga inst the whole v iru s ,

and th is is ca lled the V ir io n (V) a n tig en. I t is now g e n e ra lly agreed tha t the

V -an tigens a re the m a jo r s tru c tu ra l p ro te in s , V -a n tig e ns a re detected in

the n u c le i o f p e rm is s iv e c e lls la te in the ly t ic cycle , fo llo w in g v ir a l DNA

re p lic a t io iy b y im m u n o llu o rcsce n t s ta in ing (M ayor e t a l . , 1962). S ince i t is

l ik e ly tha t the capsid p ro te in is syn thesised in the cy top lasm , its p resence in

. 4 9

the nucleus is p ro b a b ly due to ra p id tra n s p o rt fo r v iru s assem bly. I t is

p oss ib le , how ever, tha t V -a n tis e ru m is sp e c ific fo r assem bled coat p ro te in (s )

w h ich m ay be p resen t on ly in the c e ll n u c le i (K hour y and Salzm an, 1975).

U n like the V -a n tig e n , the in t r anucle a r tu m o u r (T) antigen appears

e a r ly fo llo w in g v iru s in fe c tio n (10 - 18 hours - Rapp et al . , 1964; Hoggan

e t a l . , 1965),W hile i t was f i r s t detected us ing a com plem ent fix a tio n (CF)

method, (Takem oto and Mabel, 1965), i t can be lo c a lis e d in the nucleus

us ing im m un o fluo re sce n t (IF ) techniques (Rapp e t al . , 1964), A ntibod ies

fo r the detection of T -an tigen a re p re sen t in sera fro m an im a ls bea ring

SV40 o r po lyom a tu m o u rs . T -a n tig e n is heat la b ile ; se n s itive to try p s in

but not to D N A ’se (G ilden et a l . , 1965)' I t s syn thes is is not a ffected b y

in h ib ito rs o f DNA re p lic a tio n (G ilden et a l . , 1965) bu t is a ffected by

ac tino m yc in D, in te r fe ro n (Oxman et al . , 1967) and cyc lohex im ide (G ilden

and G arp, 1966). h l i is suggests tha t de novo tra n s c r ip t io n and tra n s la tio n

o f v i r a l DNA are re q u ire d fo r T -a n tig e n p roduc tion . C e ll- f re e tra n s la tio n

of SV40 e a r ly m essenger RNA coding fo r v i r a l T -a n tig e n (P rives e t a l. , 1977)

has led to the id e n tif ic a tio n o f SV40 T -a n tig e n (Robb, 1977) as a p ro te in w ith

a m o le c u la r w e ight o f about 9U - 100, 000. Study of SV40 host range m utants

(section 1 .4 . 1) which a re no lo n ge r able to tra n s fo rm c e lls to anchorage

independent g row th has led to the id e n tif ic a tio n of l i t t le t antigen (a p ro te in

of m o le c u la r w e ight 15 - 20, 000) (P rives et al . , 1977; C ra w fo rd e t a l . , 1978).

Recent evidence po in ts to the fa c t tha t T and t are im m u n o lo g ic a lly re la te d

and n e a r ly id e n tic a l b io c h e m ic a lly (F ie rs e t a l. , 1978), w ith t re p re se n tin g

a trunca ted fo rm of T ,

T -a n tig e n has been shown to be a p re re q u is ite fo r SV40 re p lic a tio n ;

is rec ju ired fo r the in it ia t io n and the m aintenance of tra n s fo rm a tio n ; i t is

a lso an aU toregu la to r in tha t i t shuts o ff its own syn thes is a t the le v e l o f

t ra n s c r ip t io n (F ie rs e t a l . , 1978, and re fe rences th e re in ). Both T and t

a re coded fo r in the e a r ly reg ion o f SV40 DNA (F ie rs et a l. , 1978) how ever the

appearance v a r ia b i l i t y of T -a n tig e n does depend in p a r t on host c e ll

p a ra m e te rs (Kaplan e ty a l. , 1976).

T lie U - antigen has been detected by GF and IF m ethods in SV40-

in fec ted and tra n s fo rm e d c e lls ; is found at the n u c le a r memJarane and is

heat stab le (Lew is and Rowe, 1971). U -an tigen re a c ts w ith se ru m from .

. 5 0

tu m o u r b ea rin g h a m s te rs . T and U -antigens were thought to d if fe r at le a s t

in p a rt, because a few batches o f a n ti T se ra did not conta in a n ti U antibod ies.

F u rth e rm o re an adenovirus SV40 m utant (Ad 2'^ND, ) induces U antigen but

not T antigen (K hour y and Salzm an, .1975), R ecently i t has been shown tha t

T and U antigens a re both encoded in the e a r ly re g ion of SV40 DNA and a re

now known to be the same m o le c u la r species. I t is poss ib le tha t U antigen

is in fa c t T -a n tig e n lo ca lis e d d if fe re n t ly in tra c e llu la iT y (Robb, 1977).

T u m o u r-S p e c ific T ra n sp la n ta tio n A n figenS '(TS T A ) a re the le a s t w e ll

understood of the v iru s re la te d .neo-antigens. They can be studied in v ivo

by tra n sp la n ta tio n re je c t io n (Coggin e t a l . , 1969) o r in v i t r o w here they are

lo ca lise d a t the c e ll su rface (Habel, 1961; S jogren et a l. , 1961; H abel and

Eddy, 1963; Koch and Sabin, 1963; D efendi, 1963). TSTA p ro du c tio n can be

b locked w ith in h ib ito rs of p ro te in syn thes is and D N A-dependent RNA syn thes is

suggesting tha t TS TA is a v iru s - in d u c e d p ro te in d u r in g p ro d u c tive in fe c tio n

(G ira rd i and D efend i, 1970). TS TA was p a r t ly p u r if ie d b y Chang et a l . , (1977);

i t has been found in n u c le a r p re p a ra tio n s o f SV40 tra n s fo rm e d c e lls (Anderson

et a l . , 1977) and b inds to c e llu la r DNA (Chang et a l. , 1977). T he re is a

suggested hom ology between TS TA and T -a n tig e n and th e re appear to be two

ro le s fo r th is p ro te in ,th a t o f a tu m o u r re je c tio n agent in v iv o and a re g u la to ry

p ro te in fo r the g row th of c e lls in v i t r o . (Chang e t a l . , 1977).

F in a lly , the re a re se ve ra l o th e r v iru s induced c e ll re la te d antigens

w h ich have been detected; cy to p la sm ic C -an tigen (T i lz e t^ a l. , 1969); c e ll

su rface SV40 sp e c ific g ra ft re je c tio n a n tig e n -T A T A (D uthuherbet e t a l. , 1973);

the tu m o u r associa ted c e ll su rface an tigen - TSSA w h ich is id e n tic a l w ith the

S-a n tig en detected by m em brane im m uno fluo rescence and is p ro ba b ly

id e n tic a l to TSTA antigen (Kato, 1977). These th re e , TSTA , S and TSSA

m ay in fact be fe ta l a n tigens sw itched on by the v iru s (Kato , 1977).

The in te r re la t io n s h ip s between the p o iyom a v iruse s have been

d iscussed e a r l ie r (see sections 1. 1 .2 and 1 .2 ,3 ) and at le a s t po lyom a, SV40,

BKY (REV and M M V), JCV and STM V are s im i la r a n tig e n ic a lly in some

respec ts and d if fe re n t in o th e rs .

W hile not c o n c lu s iv e ly proved in a ll SV40 p o ly o m a v iru s -c e ll sys tem s, .

i t is p robab le tha t two o r th ree m ain antigens e x is t in v iru s p e rm is s iv e o r

tra n s fo rm e d c e lls ; the V -a n tig e n ; the n uc lea r T -a n tig e n (same a s ( j ) ; arid

. 5 1 ,

the TSTA antigen ( l ik e ]y to be the same as TSSA and S antigen). I t is quite

poss ib le tha t T and TSTA antigens a re hom ologus (Chang e t a l . , 1977).

The ten ta tive conc lus ion may be tha t v iru s re p lic a tio n o r tra n s fo rm a tio n

re s u lts in two m ain an tigen ic changes. F i r s t ly v iru s components in the

nucleus, w hich appear in th e ir f in a l fo rm as V -a n tig e ns but at va rio us

in te rm e d ia te stages a re com plexed w ith o r a re id e n tic a l to foe ta l antigens

expressed as a re s u lt of the in it ia l in fe c tio n . T h a t the s itu a tio n is so

com p lexes due to '.(l) the v a r ie ty of techniques used (2) the v a ry in g degree

to which v iru s e s proceed to re p lic a tio n o f tra n s fo rm a tio n in d if fe re n t c e ll

system s (3) the use of d if fe r in g te rm s fo r the same phenomena and (4) the

in a b il ity , u n til v e ry re c e n tly , to ch a ra c te ris e the antigens be ing detected.

1 .5 .2 Human Pa p illo m a v iru s - Im mun o lo g ica l Aspects

Pass et a l. , (1971) have dem onstra ted nuc lea r and c e ll su rface

antigens in human w a rt tissues detected by in d ire c t IF us ing a n tise ra

p repa red in ra b b its to liom ogenates o f w a rt tissu e , TTiie tw o antigens w ere

iso la ted fro m tissue liomogenates by e lu tio n ' fro m antibody-sepharose im m uno-

adsorbents and n e ith e r was found to be a s tru c tu ra l com ponent of HPV. The

antigens w ere detected in w a rts , squamous c e ll ca rc in om as , fo e ta l sk in and

in p s o r ia t ic e p id e rm is , but not in basal c e ll ca rc inom as o r n o rm a l sk in

(Pass and M a rcu s , 1973). These antigens co rrespond to the T o r TSTA

antigens seen fo llo w in g in fec tio n by po iyom a v iruse s (section 1 .5 .1 ) .

V iru s antigens have also been detected, A lm e id a and c o -w o rk e rs

f i r s t observed h u m o ra l response îto HPV (A lm e ida and G offe, 1965; Goffe

e t a l. , 1966), Genner (1971) and O g ilv ie (1970) co n firm e d the presence o f

an tibod ies to V -a n tig e n but w ere unable to c o rre la te w a r t re g re s s io n w ith

antibody t i t r e us ing com plem ent f ix a tio n and passive haem agglu tina tion

techn iques. These authors re p o rte d tha t the a n ti-V antibody was la rg e ly in

the fo rm o f IgM and conside red i t in e ffe c tive in p roduc ing re g re s s io n o r

p ro te c tio n to HPV in fec tio n (Goffe el: ad ., 1966). Pass and M a ize l (1973) found

tha t anti - V antibody was d ire c te d tow ard the m a jo r s tru c tu ra l p ro te in (VPl)

of the v ir a l capsid . V -a n tig e n can be dem onstra ted in the n u c le i o f ve rru ca e

(W a lte r e t a l. , 1965). M ore re c e n tly , re g re s s io n of v e rru c a e has been

associa ted w ith an ti-V IgG im m unog lobu lins , how ever not a l l v e rru c a e conta in

. 52.

V -a n tig e n (M atthew s and S h iro d a ria , ]973; S h iro d a ria and M atthew s, 1975).

Pyrhonen and Johansson (1975) suggested tha t the cu re fo r v e rru ca e was

re la te d to IgG a n ti-V antibodies w hich they detected using im m unod iffus ion (ID )

and com p lem ent f ix a tio n (C F).

U sing ID, no antibody - antigen re a c tio n was detected us ing condylom a

e x tra c ts ,h o w e ve r c o u n te r- im m u n o e le c tro p lio re s is d id de tect anti-condyloma

antibod ies but these w ere a pp a re n tly not re la te d to condylom a re g re s s io n

(A bca rian and Sharon, 1977), N eve rthe less , the use o f autogenous vaccine

d id re s u lt in the ra p id re g re s s io n o f condylom a in 80% o f p a tien ts (A bcarian

and Sharon, 1977 ; A b ca ria n et a l. , 1976). The use o f autologouc vaccines

in the tre a tm e n t o f ju ve n ile la ryn g e a l p a p illo m a to s is has a lso m e t w ith

cons ide rab le success (Pinson et a l. , 1976).

C lin ic a l in ve s tig a tio n s re p o r t re g re ss io n of w a rts a fte r in fla m m a tio n

(Tagam i e t al . , 1974); a fte r tre a tm e n t w ith a tra n s fe r fa c to r (Stevens et a l. ,

1975); d i-n itro c h lo ro b c n z e n e (DNCB),* and a v a r ie ty o f im m une adjuvants

(L e w is , 1973; G runbe rg e ^ a l . , 1 9 7 3 ). In re n a l tra n sp la n t p a tien ts o r pa tien ts w ith long

la s tin g c e ll-m e d ia te d im m un od e fic ien c ie s ,w a rts are num erous and r a re ly

re g re s s (T liiv o le t e ^ a l . , 1975; Spencer and A nderson, 1970; Reid e t a l. , 1976;

Johansson et a l . , 1977). The increased preva lence of w a rts in pa tien ts w ith

sys tem ic lupus eryChem atosis (SLE) is in v e rs e ly c o rre la te d w ith rheum ato id

fa c to r a c t iv ity and p reva lence o f a n ti-V antibodies (Johansson et a l. , 1977),

Delayed h y p e rs e n s it iv ity sk in re a c tion s (DHR) to w a rt tissu e e x tra c ts

(M aderna, 1934) and p u r if ie d HPV have been re p o rte d . These w ere m ost

fre qu e n t in pa tien ts w ith re g re s s in g o r "p a s t" w a rts (V iac e t a l. , 1977).

The incidence of a n t i- V antibod ies was h ig he r in p a tien ts w ith a p o s itive

DHR fo llo w in g te s t, suggesting a "b o o s te r" e ffec t.

C e ll m ediated im m u n ity (CM I) is l ik e ly to be an in te g ra l p a r t o f the

host defence to w a rts (Lee and E is in g e r, 1977). C M I to HPV and tissu e

e x tra c ts can be m easured us ing lym phocyte tra n s fo rm a tio n (L T ) ( Lee and

E is in g e r, 1977) and leucocyte m ig ra t io n in h ib itio n fa c to r (L M IF ) (M o rr is o n ,

1974 and 1975b). C M I is s h o rt liv e d and could on ly be dem onstra ted in pa tien ts

w ith w a rts of less than 1 ye a r d u ra tio n . Lee and E is in g e r (1977) co n s id e r tha t'

the la c k o f c o rre la t io n o f L T and L M IF re s u lts us ing tissu e e x tra c ts com pared

w ith p u r if ie d HPV in d ica te the presence o f non -V antigens in w a r t e x tra c ts . •

. 53 .

M o rr is o n (1975a) found no c o rre la t io n between C M I and a n ti-V an tibod ies .

The ex is tence of s e ve ra l types o f HPV has been d iscussed e a r l ie r

(see section 1. 1.2 and 1.3) and P fis te r and Z u r Hausen have re c e n tly

dem onstra ted se roep idem io log 'ica l d iffe re n ces in the p reva lence of a n ti-V

antibod ies to tw o HPV ty jies (P fis te r and Z u r Hausen, 1978).

In conc lus ion , HPV is s im i la r to the p o iyom a v iruse s (Section 1,5. 1)

in p roduc ing both V -a n tig e ns and HPV-induced c e ll associa ted antigens

(T o r TS TA ).

1. 5. 3 Shope P ap illom a V i rus (SPV) - Im m unolog ic a l A spects

In v ivo and in v i t r o , SPV has been shown to produce th re e antigens.

The V -a n tig e n has been dem onstra ted w ith in p ap illom a c e lls in v ivo (Noyes

and M e llo rs , 1957), in ce lls in fected ip_vitro_ but not tra n s fo rm e d (Osato and

Ito , 1968) and pap illom a d e rive d c e lls grow n in v i t r o (Ito , 1970; S h ira to r i

e t_ a l., 1969). F u rth e rm o re , cons ide rab le evidence now supports the

presence o f SPV associa ted n u c le a r T -a n tige n s in v ivo (Z ib le r , 1958) and in

SPV in fec ted c e lls in v it ro (Yoshicla and Ito , 1968), A no th e r SPV-induced

event is the appearance of su rface antigen (Ish im o to and Ito , 1969 and 1971)

detected by TF on unfixed cu ltu re d c e lls d e rived fro m C o tto n ta il p ap illom a

tis su e . L ik e the p o iyo m a v iru se s and HPV, SPV induces both V -an tigens and

c e ll associa ted antigens (T o r TS TA ),

1 .3 .4 Bov ine Pa p illo m a V irus (BPV) - Im m uno log ica l A spects

V -a n tig e n has been detected in f ib ro p a p illo m a s us ing IF (Sm ith ies

and O lson, 1961). Q uan tita tion of BPV and a n ti-V antibody (K o ile r et a l. ,

1974) us ing im m un o d iffu s io n was used to investiga te the re g re s s io n of

f ib ro p a p illo m a s in re la t io n to a n t i-V antibody in the se ru m . No c o rre la t io n

was found between fib ro m a re g re s s io n and a n ti-V antibody and fib ro p a p illo m a s

continued to g ro w p ro g e s s iv e ly (Bartho ld and O lson, 1974a).

The f ib ro m a ce lls of BPV induced fib ro p a p illo m a s la c k V -an tigens

de tec tib le by IF and F M (S m ith ies and Olson, 1961; Ta juna e t a l, , 1968;

Robl and O lson, 1968) but possess a su rface antigen detected by in d ire c t IF

on liv e un fixe d BPV induced fib rom a ce lls g row n i n v iv o and in v i t r o . (Bartho ld

and O lson, 1974b). S e r ia lly sam pled fib ro p a p illo m a s show cons ide rab le

. 54 .

h is to p a ü io lo g ica i evidence of a m a jo r ro le played by C M I in the re g re s s io n

o f at le a s t the f ib ro m a com ponent o f bovine f ib ro p a p illo m a s (Bartho ld and O lson,

1974b).

Bovine p a p illo m a v iru s is thus s im i la r to SPV, HPV and the po iyom a

v iru s e s in its capac ity to induce V -a n tig e n and c e ll assoc ia ted antigens.

A lthough no n uc lea r T -a n tig e n has been dem onstra ted in BPV induced tum ours ,

th is m ay be due to in s u ff ic ie n t re s e a rc h since these have been found in HPV

and SPV in fected c e lls .

1 .5 .5 O ther P ap illom av iruse s - Im m un o lo g ica l Aspects

A V -a n tig e n has been detected in canine o ra l p ap il lo m a s and a n t i-V

antibody in dogs w ith re g re s s in g tum ours and the lik e lih o o d of a c e ll associated

n o n v ir io n antigen is d iscussed by Cham bers e t a l. , (1960). O the r

p a p illo m a v iru s e s have not been stud ied in s u ff ic ie n t depth to p ro v ide usefu l

co m p ara tive data.

1 .5 .6 S um m ary - Im m uno lo g ica l A spects of P apovaviridae

The response of the host to papovavirus in fe c tio n is com p lex and

s t i l l p o o r ly understood. I t d o u b tle ss ly invo lves at le a s t th re e m a in defence

m echan ism s: a n t i- v iru s antibody; a n t i- c e ll antibody and c e ll m ediated

im m u n ity . The re la t iv e c o n tr ib u tio n of each is unknown a lthough m ost authors

postu la te tha t a ll th re e a re re q u ire d fo r e ffe c tive re je c t io n of e x is tin g

in fe c tio n and re s is ta n ce to challenge.

1. 6 Physicochem ica l Ch a ra c te r is t ics o f the Papo v a v ir id a e

M ention has been made in e a r l ie r sections o f th is re v ie w of the

chem ica l com pos ition of papovaviruses and these fea tu res w i l l not be d iscussed

in g re a t d e ta il he re . T h ree phys icochem ica l aspects a re how ever, im p o rta n t.

They a re : (1) a b i l i ty to agg lu tina te re d blood c e lls ; (2) v iru s p a r t ic le

d ia m e te r, and (3) v iru s th e rm o s ta b ility . T h e ir im po rtance is due to the fa c t

tha t d iffe re n ce s in these ch a ra c te rs a re often used to d iffe re n tia te between

in d iv id u a l papovav irus m em bers . Indeed, v iru s s ize and the re la te d

p a ra m e te rs o f v iru s and DNA m o le c u la r w e ight a re one o f the m a in ch a ra c te rs

used to separate the p o iyom a v iruse s fro m the p a p illo m a v iru s e s . T h e rm o - ■

55

s ta b il i ty is o f im po rtance , e s p e c ia lly w ith BPV, s ince m .ilk fo r human

consum ption is l ik e ly to be contam inated w ith BPV fro m tea t p ap illom a ta .

H aem agg lu tina tion lias u n til v e ry re c e n tly rep resen ted one o f the p r in c ip a l

m ethods of q ua n tita tin g v iru s and a n ti-V an tiliod ie s .

1 .6 .1 P apovav ir u J I a em agg lu tinatio n

P olyoma v i r u s agg lu tina tes re d blood c e lls (RBC) o f m any species

(Sachs e ^ M . , 1959) but guinea p ig RBC are com m on ly used. I t attaches

to RBC by the same mu co p ro te in re c e p to rs as in fluenza v iru s . These

re ce p to rs can be destroyed b y in fluenza v iru s o r re c e p to r d e s tro y in g enzyme

(RDE) fro m V ib r io cony mm but po lyom a its e lf does not d e s tro y the re ce p to rs

(H a rtle y e t a l. , 1959). T re a tm e n t of tissue cu ltu res w ith RDE reduces th e ir

s u s c e p tib ility to po lyom a v iru s in fec tio n suggestions th a t the re ce p to rs a re

invo lved in the a ttachm ent o r e n try of v iru s in to c e lls (Rowe, 1961). The

haem agg lu tin in is p resen t on both fu l l and em p ty p a r tic le s and is re a d ily

e luted fro m the e ry th ro c y te re ce p to rs w ithou t d e s tro y in g them at 37^C

(C raw fo rd , 1969). The haem agg lu tina tion assay fo r v iru s o r antibody m ust

th e re fo re be c a rr ie d out a t 4^C (Rowson, 1976).

The K -v iru s o f m ice haem agglu tina tes sheep RBC a t room tem pe ra tu re

o r 37°C . H eating of the v iru s suspension at t im e s unm asks the haem agg lu tin in

(K ilh a m , 1961).

RKV o f ra b b its agg lu tina tes guinea p ig RBC at 4°C and 20°C and lik e

po lyom a, re c e p to rs are destroyed by RDE (H a rtle y and Rowe, 1964).

pap illo n m v i ru s does not haem aggluntinate RBC, how ever ra b b it

RBC do adsorb the v iru s (Andrewes and P e re ira , 1972).

W hile SV40 does not agg lu tina te RBC, the c lo s e ly re la te d BKV does

agg lu tina te human, guinea p ig and ch icken RBC (G ardne r e t a l . , 1971;

M a n ty ja rv i e t^ a l. , 1973). M ax im um HA is achieved w ith human O RBC at

4°C in b u ffe rs p l l6 , 1 - 7. 4 w ith t i t r e s about fo u rfo ld g re a te r than w ith guinea

p ig RBC (M a n ty ja rv i et_aT , 1973; S eehafe r c t al . , 1975). The v iru s

associated haem agg lu tin in is re s is ta n t to e th e r; heating a t 560 C fo r 30 m inu tes ;

c h lo ro fo rm ; and 0.1% p - p ro p io la c ton e . No MA is observed at 37^C

(D ougherty and D iS tefano, 1974; P itko ef a l. , 1975; Takem oto and M u lla rk e y ,

1973). The human RBC HA re ce p to rs w ere reduced but not destroyed by RDE,

. 56,

The in h ib ito rs o f BKV HA are p re sen t in se ra o f m any species and these m ay

be rem oved us ing RDH, acetone and sodium iod ide (Padgett and W a lke r, 1976),

L ik e BKV, JC V agg lu tina tes human, guinea p ig and ch icken RBC at

4°C (Padgett and W a lke r, 1973; W a lke r et al . , 1974). The a g g lu tin in is

v iru s associa ted (Osborn e t_ a l., 1974). Sera o f m any species conta in HA

in h ib ito rs and sod ium iodide is used to rem ove them (Padgett and W a lke r,

1973).

BPV has been shown to agg lu tina te mouse RBC at 4bC between pH6. 8

and 8. 4, BPV is e lu ted a t 37°C lik e po lyom a and BKV. RBC re c e p to rs are

re s is ta n t to RDE and In fluenza neu ram in idase . HA is assoc ia ted w ith both

fu l l and em p ty p a r tic le s (F avre et a l . , 1974).

1. 6. 2 Papovav i r u s Par t ic le D ia m e te r

Tab le 11 l is ts the wide, range of e s tim a te s o f v iru s p a r t ic le d ia m e te r

us ing both n e g a tive ly sta ined and th in section e le c tro n m ic ro s c o p y . I t should

be noted tha t each technique has its ' own b ias . N e g a tive ly sta ined

p re p a ra tio n s m ay conta in fla ttened v ir io n s and a lso a p ro te in coat m ay on

occasions lead to a la rg e r apparent s ize . T h in section d ia m te r es tim a tes

m ay be s m a lle r because of app rec iab le shrhrkage due to deh yd ra tion and

em bedding (G ross, 1970).

The e s tim a te s v a ry co n s id e ra b ly w ith the fo llo w in g ranges a cco rd in g to

Tab le 11: P o lyom a- 2mp; SV40 - 5mp; BKV- 3. 5mp; JC V -5 . 5mp; K - lOmp;

STM V - 5mp; HPV- 27mp; SPV- 34mp; ROPV- 12mp; Canine OPV- lOmp;

D e e r F V - 18mp; BPV- 19mp. W liile the re are w ide d iffe re n ce s l ik e ly to be

caused by p re p a ra to ry m ethods (see e a r l ie r ) i t is s u rp r is in g to notice tha t

the v a r ia t io n in d ia m e te r es tim a tes o f the p o lyom a v irus g roup range 2 - lOmp

and the p a p illo m a v iru s g roup range 10 - 34mp. M ention has a lre a d y been

made of the fa c t tha t d if fe re n t types of HPV occur (sec tion 1. 3) and though no

evidence appears in the li te ra tu re , i t m ay be tha t d ia m e te r m easurem ents o f

s ing le tyiaes w i l l be d if fe re n t to o th e rs . In th is context the p o iyom av iruses

of m onkeys, STM V, OMPV and SV40 are fro m 30 - 45 mp, a range o f 15mp;

a lte rn a t iv e ly the p o iyom a v iruse s o f m ice , po lyom a and K -v iru s a re fro m

40 - 50mp, a range of lOmp.

In s u m m a ry , i f one c la s s if ie s papovaviruses acco rd in g to species of

, 5 7 ,

TABLE 11 : PAPOVAVIRUS PARTICLE DIAMETER,

V iru s PrcpK D ia m e te r (m p) R eferences

Polyoma U 45 (1); 43 (2) l.A n d re w e s and P e re ira , 1972

SV40 N 42.61 (3) 2 . Rowson, 1976

U 40-45 (4) 3 .B ahr et a l . , 1976

BKV N 3 8 -4 3 .5 (5) 4 .A n d e re r e t a l . , 1967

JCV N 3 8 -4 3 .5 (5) 5 .Padgett and W a lke r, 1976

T 39.1 (5) ô .R e is s ig e t a l , , 1976

K -v iru s U 40-50 (1) 7 .D a n ie l et a l . , 1976

RKV T 47 (1) 8 .M e ln ic k et a l . , 1974

STM V N 40-45 (6) 9 .W ill ia m s et a l . , 1961

OMPV T 33 (7) 10. M e ln ick et a l . , 1952

HPV N 55 (8); 55 (9); 52 (10). 11. G ross , 1970

T 40-54(ll);33(12);38(13) 12. C h a rle s , 1960

HPV (E'EH) T 40-60 (14) 13. Bunting, 1953

SPV N 56-60 (1.5); 40-47 (11) 14.P ra e to riu s -C la u se n andW il l is , 1971

T 26-29 (16); 33 (17) 15 .C ham bers and Ito , 1964

ROPV N 50-52 (18) 16.Haguenan et a l , , 1960

T 40 (18) 17 .M oore et a l . , 1959

Canine OPV N 40-50 (19) 18.Rozok et a l . , 1966

D eer F V N 50-53 (20) 19. C h e v ille and O lson, 1964a

T 35 (20) 2 0 .T a jim a e t a l . , 1968

FPV N 51.7± 1.7 (21) 21.A Ib e r t e t a l . , 1977

BPV N 47.5 (22); 47(23);51-54(20) 22 .H uck , 1965

T 35 (20) 23. L e vy et a l . , 1963

Prep - P re p a ra to ry method - N - negative s ta in ing ; T = th in section ;

U = u nsp e c ifie d ,

58

o r ig in , p a r t ic le d ia m e te r e s tim a tes range, in the polyom a group 3 .5 - 15mp

and the p ap illom a group 10 - 34m p. The p ra c tic e of d if fe re n tia t in g the

p apovav iruses on v iru s size g iv in g p o iyo m a v iru se s a gene ra l range 43 - 45mp

and p a p illo m a v iru s e s 52 - 54mp (Andrewes and P e re ira , 1972) is not

supported by the l i te ra tu re w h ich shows subs tan tia l ove rlap between the

g ro up s .

1 .6 .3 T h e rm o s ta b ility o f Papovaviruses

Tab le 12 su m m a rise s the lite ra tu re on the th e rm o s ta b ility o f the

p a p o va v ir id a e . M ost m em bers a re able to su rv ive a t leas t 30 m inu tes at

60°C , the equ iva len t of p a s te u r is a tio n . I t is o f in te re s t tha t the DNA is able

to w ithstand iiigh te m p e ra tu re s fo r long pe riod s la rg e ly because o f i t s '

unique^closed, c ir c u la r fo rm . A t h igh tem pe ra tu res the DNA d isassoc ia tes

in to s ing le s trands but reassoc ia tes on coo ling (C raw fo rd , 1964). The

increased th e rm o s ta b ility o f v i r a l DNA over whole v iru s m ay be due to the

e a r l ie r dena tu ring o f v i r a l capsid p ro te in .

T h e re is no re fe re n ce in the l i te ra tu re on the e ffe c ts o f t im e -

te m p e ra tu re on BPV. T h is thes is w i l l re p o r t on th is aspect because of its

p o te n tia l im po rtance in th ree a reas (1) a n im a l to a n im a l tra n s m is s io n of teat

p a p illo m a to s is v ia heat trea ted m ilk in g devices (2) adu lt to c a lf tra n s m is s io n

using pas teu rised m ilk (3) human exposure to BPV v ia p as teu rised m ilk .

1 .7 S um m ary o f the L ite ra tu re Review

T ab le 13 su m m a rise s the m a in e ffec ts o f the papovav iridae in v ivo ;

Tab le 14, the m ain in v it ro e ffe c ts ; and Table 15, im m iu io lo g ic a l and

p h ys ico ch e m ica l c h a ra c te r is t ic s . W h ile m ore re se a rch is needed, i t is

apparent that in m ost c h a ra c te r is t ic s , the polyom a and p a p illo m a v iru s e s

overlap co ns ide ra b ly , often m aking the separa tion o f the papovav iridae in to

two groups m ore confus ing than h e lp fu l. T iie m a jo r d iffe re n ce re m a in in g

between the two g roups is that the re is a t p re sen t no re lia b le method of

g ro w in g p a p illo m a v iru s e s in v i t r o . I t is p robab le that th is is due to the

absence o f a su itab le c e il system , since some p o iyo m a v iru se s have

e x tre m e ly re s t r ic t iv e g row th re q u ire m e n ts .

, 59 .

TABLE 12: TIME:-TEMPERATURE INACnVATïON OF PAPOVAVIRUSES

V iru s T im e (m in ) T em p e r a t u r e C ) E ffec t* R eferences

SPV 30 67 N I ) F is c h e r and Green,30 70 I ) 1947

ROPV 30 65 N I ) Parsons and K idd,30 70 N I 1943

HPV 30 50 N I Rowson and Mahy,1967

Canine O ra l 60 45 N I ) A nd rew es andPV 60 58 I I P e r ie ra , 1972

Equine Skin 30 55 I ) Cook and O lson,PV ) 1951

Polyom a 30 60 NT . ) B radsky et a l , ,30 70 I ) 1959

K -v iru s 180 70 N I ) F is c h e r and270 70 I I K ilh a m , 1953240 60 N I )

SV40 60 46 N I M a yo r et a l . , 1963

RKV 30 60 N I ) H a r t le y and Rowe,30 70 N I ) 1964

JCV 60 50 N I Padgett et a l , , 1977

SPV DNA 80 70 N I ) Ito , 1975180 70 N I )360 70 N I )

E ffe c t: N II

not inactiva ted in a c tiva te d .

.60

TABLE 13: THE PAPOVAVIRIDAE IN VIVO

T um our type Benign Benign C arc inom a Adenoma Sarcom a O the rE p ith e lia l M esenchym al

VIRUS

Polyom a + -h d- d- d-

SV40 - + - +

BKV - T - + d-

JCV - + - - +

STM V - - - -

OMPV - - - - -

HDV " - - - -

K -v iru s - - - - -

R KV - - - *' -

HPV H- - d- - -

SPV + - + - -

ROPV + - - “ -

Canine PV + - - - -

Canine OPV + - - -

Equine PV + - - - -

Goat PV + “ + - ~

H a m ste r PV + d- d- - T

Sheep PV d- d- ~ - d-

D ee r F V H- d- “ - d-

Equine S arco id T d- - - -

BPV T + -h d-

N ote: T re p o rte d to occur in the lite ra tu re

+

T

+

+

not re p o rte d in the l i te ra tu re

.61.

TABLE 14: THE PAPOVAVIRIDAE IN VITRO

V iru s w i l l in fec t w i l l re p lic a te w i l l tra n s fo rm

Polyom a + H- d-

SV40 + +

BKV -1- T +

JCV + d- d- / -

K “ V iru s H- d-

RKV + + / "

HD v iru s + + d-

STM V T d- -

HPV -f / - d-

SPV + + / "

BPV -f + / “ d-

O the r PV

Note: + = re p o rte d in the li te ra tu re

= not rep o rte d in the lite ra tu re

+ / - - uncon firm ed re p o r t (s) in the l i te ra tu re .

. 62 .

TA B LE 15: IM M U NO LO G IC A L AND PHYSICO CHEM ICAL CHARACTERISTICSOF PAPOVAVIRUSES ’

V iru s V -an tigen n u c le a r c e ll m em brane H .A . D ia m e te r T h e rm o antigen antigen (mp) s ta b il ity

Polyom a -f- d- + + 43-45 d-

SV4Ü + d- d- 40-45 T

BKV + d- d- + 4 0 .5 -4 4 -

JCV + d- + d- 3 8 -4 3 .5 +

K -v iru s - “ - d- 40-50 d-

RKV “ - + 47 +

STM V - - - - 40-45 -

OMPV - - " 33 -

HPV d- d- d- - 33-60 -

SPV d- d- d- + / " 26-60 d-

BPV + T d- 35-54

Canine OPV d- - + / - “ 40-50 "

D e e r F V - - - “ 50-53 -

ROPV - - - “ 40-52 d-

FPV - - “ - 50 -5 3 .5 -

Equine PV - - - - - -

+ - p re se n t o r p o s itiv e

- not re p o rte d o r negative

+ / " - p oss ib le o r p robab le

- v i r u s s u r v i v e s h e a t i n g a t 6 0 ^0 f o r 30 m in u te s

. 63.

The l i te ra tu re rev iew ind ica tes the g re a t rese a rch e ffo r t tha t has been

made d u ring the la s t five ye a rs - the next five yea rs m ay w e ll re n d e r a la rg e

am ount o f c u rre n t specu la tion as m isgu ided and obso le te .

1 .8 Gen e ra 1 In tro d u c ti on

Bovine p a p illo m a v iru s n o rm a lly causes benign tu m o u rs in ca ttle and

is o f l i t t le econom ic consequence. The m a jo r loss in non -b ra cke n a reas is

re la te d to the damage to h ides and sk in s . O ccas iona l ou tb reaks can occur

w here the e ffec ts a rc severe , e sp e c ia lly so in the case o f anogen ita l f ib ro -

p a p illo m a to s is causing reduced re p ro d u c tive p e rfo rm an ce and o ccas ion a lly

death, due to o bs truc tion caused by the tum ou r o r due to secondary in fe c tio n .

E p id e m io lo g ic a lly , c a ttle a re , un like man, iso la ted w ith in in d iv id u a l herds

fo r the g re a te r p e rio d o f th e ir liv e s .

When BPV is endem ic in a herd , i t is thought tha t the c lose contact

a ffo rded by w in te r housing re s u lts in tra n sm iss io n to suscep tib le younger

a n im a ls . F ib ro p a p illo m a s on the skin th e re fo re u su a lly appear in Spring on

a n te rloven tra l sk in s ite s o f a n im a ls up to 2 ye a rs o f age (Bagdonas and O lson,

1953). Wiien a herd has been fre e o f BPV fo r a num ber o f ye a rs , the

in tro d u c tio n o f a new a n im a l m ay p re c ip ita te an outb reak of f ib ro p a p illo m a to s is

a ffe c tin g a n im a ls o f a l l ages, som etim es w ith severe econom ic consequences.

A d d it io n a lly , n o rm a l v e te r in a ry p ro ced u re s such as p regnancy tes tin g and

dehorn ing can lead to severe outb reaks In suscep tib le he rds (Tweddle and

W hite , 1977).

A m a jo r cause of econom ic loss occu rs in a re as w he re bracken fe rn

and re la ted species (1 'te r i d ium a q u ilin um , escu lent urn, Che i Ian the s s ie b e ri

and te n u ifo lia ) is p re va le n t (M cKenzie , 1978). The in te ra c tio n between

bracken and BPV is u nc lea r but some au thors have suggested a co -ca rc in og e n ic

e ffe c t - the p ro longed intake o f bracken leading to reduced im m unocom petence

a llo w in g BPV induced les ions o f the u r in a ry b ladder and upper a lim e n ta ry

t ra c t to proceed to m a lignancy ( ja r re t t et a l . , 1978 and re fe ren ce s th e re in ).

The absence of re p o r ls o f BPV-re In ted m a lignan t tum ou rs on the skin o r o the r

m ucosal su rfaces in ca ttle fro m bracken a reas ind ica tes tha t o th e r fa c to rs

m ay be in vo lved . Such fa c to rs inc lude v a r ia b i l i ty in im m une s u rve illa n ce

a t d if fe re n t s ites and the existence o f d iffe re n t s tra in s o r types of BPV. •

. 64 .

BPV is a lso im p o rta n t as a m odel fo r pap illom a v iru s in fec tio n s o f

o th e r species, in c lu d in g m an. A p a r t fro m the unresolved question of w he the r

BPV can i ts e lf in fe c t man (see F re n z , 1941; Schu ltz , 1908; G re n ie r et a l . ,

1976; P ere l and L um pk in , 1976., Rowson and M ahy, 1967), BPV does have

a w id e r host s p e c if ic ity (Olson et a l . , 1969) than the o the r p a p illo m a v iru s e s .

I t has been re la te d to the deve lopm ent o f benign and m a lignan t tum ou rs under

n a tu ra l co nd itio n s . The study o f BPV m ay th e re fo re lead to use fu l techniques

and obse rva tions a pp licab le to o the r p a p illo m a v iru s in fe c tio n s .

T h is thes is w i l l re p o r t the re s u lts o f in ves tiga tions in to the in v it ro

and in v ivo e ffe c ts of BPV and BPV tra n s fo rm e d c e lls ; the use o f BPV in v it ro

tra n s fo rm a tio n of c e lls in the developm ent o f a quanta l assay fo r BPV and

an ti-B P V tra n s fo rm e d c e ll an tibod ies , the re s u lts o f an a b a tto ir su rvey

coupled w ith e xp e rim e n ta l v iru s tra n s m is s io n and im m u n o lo g ica l re s u lts .

suggesting the ex is tence of se v e ra l types of BPV; and the e ffe c ts o f

p a s te u r is a tio n on BPV in v ivo and in v i t r o . R eports o f aspects o f the w o rk

in th is thes is w i l l be pub lished e lsew here and the re a d e r is re fe r re d to the

re fe re n ce s lis te d a t the end o f th is thes is (M eischke, 1978 a - e).

65 .

C h a p te r 2. IN VIVO E FFE C TS OF BOVINE P A P IL L O m VIRUS: A N ABATTO IR SURVEY OF BOVINE PAPILLOMATOSIS AND COMPARISON, BY E LE C TR O N MICROSCOPY AND ELECTROPHORESIS OF BPV IS O LA TE D FROM VARIOUS LESION TYPES..

2 .1 In trod u c tion

Bovine p ap illom a and fib ro p a p illo rn a a re benign tum ours w h ich o ccu r a t

m any d if fe re n t ana tom ica l s ite s . The common cutaneous f ib ro p a p illo rn a , m ost

fre q u e n tly found on a n te r io -v e n tra l skin s ite s (F renz , 1941; Bagdonas and O lson,

1953) is a s e lf l im it in g d isease of young c a tt le , F ib ro p a p illo m a s of the penis

and va g ina l m ucosa (M cEntee, 1951) a lso occur and m ay be vene re a lly

tra n s m itte d in a s im i la r fashion to the human gen ita l w a r t , condylom a

acum ina tum (Delap et a l . , 1976). P ap illom as and f ib ro p a p illo m a s o f the

upper a lim e n ta ry t ra c t and the tea ts have been re p o rte d e a r l ie r (Delap et a l , ,

1976; M a rtin et a l . , 1972)ând J a rre tt et a l . , (1978a) have shown a 19%

p reva lence of a lim e n ta ry p ap illom as in a la rg e a b a tto ir s u rv e y . The

d em onstra tion o f bovine p ap illom a v iru s (BPV) in both tissu e e x tra c ts and

sections ( ja r r e t t c t a l . , 1978a) led to the suggestion tha t a p o ss ib le source of

v iru s causing a lim e n ta ry p a p illo m a s was teat p a p illo m a to s is (J a rre tt et a l , ,

1978b), F requen t obse rva tions appear in the l ite ra tu re o f upper re s p ira to ry

and a lim e n ta ry t ra c t p a p illo m a s in ch ild re n whose m o the r had been in fected w ith

condylom a acum ina tum d u r in g p regnancy (W allenborn , 1976). G issm ann et a l . ,

(1977) have d is tingu ished fo u r d iffe re n t types o f human p a p illo m a v iru s (HPV)

and pos tu la te tha t fu r th e r types o f HPV could e x is t in condylom a acum inata and

la ry n g e a l p a p illo m a s as w e ll as some skin w a r ts .

T h is p a r t re p o r ts the re s u lts o f a su rvey o f 721 c a ttle y ie ld in g 2, 427

p a p illo m a s and com parison o f va rio u s skin and tea t v iru s iso la tes by the

e le c tron m ic rosco p e and p o ly a c ry la m id e gel e le c tro p h o re s is ,

2 .2 M a te r ia ls and M ethods

2 .2 ,1 C a ttle Survey

A t a p p ro x im a te ly w eek ly in te rv a ls , a l l c a ttle k ille d on one day a t

G lasgow a b a tto ir w e re exam ined fo r the presence of p a p illo m a on a l l s ite s of

the in tegum ent. The s ite (s ) o f any p ap illom as as w e ll as the sex and p a r ity .

. 66 ,

w e re reco rded fo r each a n im a l. E v e ry a ffected teat, sc ro tum o r p iece of

skin was rem oved and exam ined the same day in the la b o ra to ry . Skin and

tea t sam ples w e re kept in separate co n ta in e rs . In d iv id u a l tea t p ap illom as

w e re rem oved a fte r c la s s if ic a tio n in to three m a cro sco p ic types and stored

se pa ra te ly in P B S /G lyce ro l held a t 4 ^ 0 . The loca tion , num ber and

c la s s if ic a t io n o f each pap illom a was docum ented, A num ber o f p ap illom as

fro m each m a cro sco p ic ca tegory w e re sam pled, p laced in 10% fo rm o l sa line

and g lu ta ra ldehyde f ix a tiv e s fo r fu r th e r h is topa tho log ic and e le c tro n

m ic ro s c o p ic (EM ) e xam ina tion ,

2 .2 . 2 V iru s E x tra c t io n

Pooled and s ing le case p a p illo m a s w ere hom ogenised using a Si Ive rson

b lende r in to a 10% W /V crude suspension in phosphate bu ffe red sa line (PBS).

P u rifie d v iru s was p repa red a cco rd in g to m ethods described e a r l ie r

(L a nca s te r et a l . , 1976) but w ith o u t the use o f de te rgen t o r u ltrasound to a id

d is p e rs io n .

2 .2 .3 P ap ill oma C la s s ific a tio n

A l l teat p ap illom as w ere ca tegorised into the fo llo v /in g types based on

g ro ss appearance. A . F rond type - these acum inate le s io n s had m u lt ip le

f i l i f o r m p ro je c tio n s o f a d e lica te te x tu re longer than 2mm and w ith o r w ith o u t

a fib ro m a tous base. P ap illom as w ith p ro je c tio n s < 2mm w e re p laced in to

one o f the fo llo w in g ca tego ries (F ig u re 2). B. F la t and Round tyipe - f ib r o

p a p illo m a s w ith a fib ro m a to u s base, often covered w ith a pp a re n tly n o rm a l

e p ith e liu m but som etim es w ith h y p e rk e ra to tic o r p a p illom a to us e p ith e liu m

w ith f i l i f o r m p ro je c tio n s < 2mm long (F igu re 3). C . R ice G ra in les ions

o ccu rre d as foca l a reas of p ap illom a tous e p ith e liu m ra re ly > 1cm d ia m e te r

(F ig u re 4 ). No m a cro sco p ic f i l i f o r m p ro je c tio n s and no fib ro m a to u s in v o lv e

m ent w e re v is ib le . In m u lt ip le cases these les ions coalesced to fo rm la rg e

m asses of pap illom a to us tissu e , o ccas ion a lly co ve ring the w ho le te a t,

2 .2 .4 E le c tro n M ic ro sco p ic E xam ination

A l l crude suspensions fro m tea t p ap illom a w ere neg a tive ly stained w ith

phosphotim gstic ac id (PTA) and exam ined fo r the p resence o f BPV. P u rifie d

. 6 7 ,

4

F ig u re 2: T ea t p ap illom a of the Frond type - no tice d e lica te f i l i f o r m

p ro je c tio n s > 2mm in leng th . Bar = 2cm .

6 8 ,

F ig u re 3; T e a t p a p illo m a of the F la t and Round type - no tice absence of

f i l i f o r m p ro je c tio n s > 2mm in leng th . H is to lo g ic a lly (not shown)

th is les ion was a f ib ro p a p illo m a . Bar = 2cm .

69 .

%

F ig u re 4: Tear p a p i l lo m a to s is of tiie Rice G ra in type . IT e s e les ions a re

m a c ro s c o p ic a l ly and m ic ro s c o p ic a l ly s im i la r to foca l e p ithe l ia l

h ype ip la s ia in m an. Note absence of f i l i f o r m p ro je c t io n s and

the d is c re te ep ithe l ia l nature of the les ions . Bar = 2cm.

70

p re p a ra tio n s w ere s im i la r ly exam ined both to c o n firm the presence o f BPV

and to su b je c tiv e ly assess the p u r ity of in d iv id u a l p re p a ra tio n s . A m in im um

of 10 e le c tro n -m ic ro g ra p h s w ere taken o f each p re pa ra tio n fo r fu r th e r

a n a ly s is .

2 .2 .5 V iru s M easurem ent

E le c tro n m ic ro g ra p h s o f a standard 3,160 lin e s /m m g rid taken

c o n c u rre n tly and a t the same ind ica ted m a g n ifica tio n as e le c tro m ic ro g ra p h s

o f p u r if ie d v iru s p re p a ra tio n s w e re m easured under a L e itz s u rg ic a l

m ic ro s c o p e . The mean d istance between the g r id lin e s o f e le c tro n -

m ic ro g ra p h s was d iv ided by the known d istance between the g r id lin e s and

the specim en to g ive the ac tua l m a g n ifica tio n . In d iv id u a l p a r t ic le s w ere

m easured fro m a m in im um of f iv e d if fe re n t e lec tron m ic ro g ra p h s . Two

d ia m e te r m easurem ents a t r ig h t angles to each o th e r w e re made o f each

v iru s p a r t ic le . O n ly " f u l l " p a r t ic le s w ere m easured . C are was taken to

inc lude equal num bers o f p a r t ic le s fro m d a rk and lig h t backgrounds in an

a ttem p t to equa lise d if fe re n t ia l s ta in p e n e tra tio n .

P u rifie d v iru s sam ples fro m the same case w e re p repa red at d if fe re n t

t im e s to de tect any t im e o r p u r if ic a t io n e ffec ts on p a r t ic le d ia m e te r.

2 .2 .6 P o lyac ry lam id e Gel E le c tro p h o re s is

The am ount of p ro te in in each p u r if ie d v iru s sam ple was de te rm ined

a cco rd in g to the method o f L o w ry . Samples fo r e le c tro p h o re s is w ere

d isso lved in sam ple b u ffe r {0.0625 M T r is .H C lp H 6 .8 , 10% g ly c e ro l, 2% SDS,

0,01% brom opheno l b lue, 5% B m ercap toe thano l) and bo iled fo r th ree m in u te s .

The cooled sam ples w ere then app lied to d iscontinuous SDS - p o lya c ry la m id e

slab ge ls p repa red a cco rd in g to the method of L a e m m li (1970). Separa ting

ge ls contained c ith e r 8% o r 11% a c ry la m id e . A p p ro x im a te ly 20 mg of p ro te in

was app lied to each w e ll. A constant p o te n tia l o f 50V was app lied d u ring

s tack ing , and th is was increased to lOOV fo r the re m a in d e r of the ru n .

E le c tro p h o re s is was stopped when the tra c k e r dye was a p p ro x im a te ly 1cm

fro m the bottom of the g e l. P ro te ins w e re v isu a lize d by s ta in in g w ith

C rom ass ie Blue R ,

71

2 . 2 . 7 Papilloma Scraping

Tw enty ind iv id ua l p ap illom as selected at random fro m each

c la s s if ic a tio n group w ere scraped w ith a s te r ile sca lpe l blade tak ing care that

on ly e p ith e lia l tissue was being rem oved. Scrapings w ere p laced in 1 m l

T r is sa line w ith EDTA and a fte r gentle ag ita tion w e re subjected to th re e

freeze -thaw cyc le s . D u rin g each cyc le the sam ple was held - 20°C fo r

ten m inu tes , thawed in a 37^C w a te r bath fo r ten m inu tes and gen tly ag ita ted .

A f te r com p le tion o f the th ird cycle the sam ple was n eg a tive ly stained w ith

PTA and exam ined fo r the presence of BPV, Samples w e re regarded as

negative i f BPV could not be seen a fte r fiftee n m inu tes sea rch ing o f a t least

two separate p re p a ra tio n s . Those pap illom a w h ich y ie lded negative sam ples

w e re re -s a m p le d on a d if fe re n t s ite and re -exam ined im der the e le c tron

m ic ro s c o p e .

2 ,3 R esu lts

A l l ca ttle k ille d on each o f f ive days w ere trea ted as separa te sub

su rve ys . Tab le 16 su m m a rise s the re s u lts of the to ta l su rvey (Appendix A

conta ins the de ta iled re s u lts o f the su rve y ). Standard dev ia tions w ere

ca lcu la ted using the five subsurvey means to tes t the s ig n ifican ce of d iffe re n ce s

between groups as lis te d o n T ib le 17 using the Students t - te s t . 2 0 .8± 5,8% of

m ale ca ttle ; 4 8 .2± 3.7% o f parous and 31.0± 7,5% of n u llip a ro u s fem ale

c a ttle had teat p a p illo m a to s is . The d iffe re n ce s between m a le ca ttle and

fem ale c a ttle w e re s ig n if ic a n t. The average num ber o f tea ts w ith p ap illom as

p e r a ffected a n im a l was 1,4± 0 .3 in m ale ca ttle ; 2 .3 ± 0.1 in parous and

2.1+ 0 .3 in n u llip a ro u s fem ale c a tt le . These d iffe re n ce s w e re s ig n if ic a n t.

T hroughout the whole su rvey 36% of ca ttle had teat p a p illo m a to s is , each

a ffected a n im a l had a p p ro x im a te ly two teats w ith p a p illo m a s on them .

Tab le 18 shows the p ro p o rtio n o f the th ree pap illom a types in a

to ta l o f 2 ,427 p a p illo m a s found in th is su rve y . A p p ro x im a te ly h a lf

(5 2 .7± 12.4%) w ere r ic e g ra in les ions (R G )j26 .9± 8,2% w e re of the frond type •

(F ) and 20 .4+ 5.9% w ere o f the f la t and round type (F R ). An average of

5.1± 1 p a p illo m a s p e r a ffected teat is re p o rte d .

. 72 .

TABLE 16: Number of C attle and T ea ts affected with Papillom atosis

Type o f A n im a l* M C H MGH

A n im a ls Exam ined 167 328 226 721

A n im a ls A ffec ted 35 257 68 260

Teats A ffec ted 49 365 141 555

% A n im a ls A ffec ted ± S .D . 2 0 .8± 5.3% 48.2+ 3.7% 31 .0+ 7.5% 36%

A ffe c ted T e a ts / ± S .D A ffec ted A n im a l

1 .4 ± 0 .3 2 .3 ± 0.1 2 .1 + 0 .3 2 .1 + 0 .2

* M - a l l m .ile an im a ls - b u lls o r cas tra tes

C - cows - a l l parous fem a le a n im a ls

H ~ h e ife rs - a l l n u llip a ro u s fem ale a n im a ls .

TABLE 17: t - te s t o f mean d iffe re n ce s in % an im a ls a ffected and a ffected teats p e r a ffected a n im a l.

num ber o f

M /c 4 G /h + » /M +

D iffe re nce in % ca ttle w ith tea t p ap illom a

t-v a lu e 5 .97 1.25 3 .9 9

degrees o f freedom 8 8 8

s ig n ifican ce n .s .

D iffe re n ce in num ber affected teats p e r a ffected a n im a l

t-v a lu e 9.52 4.61 2 .4 9

degrees o f freedom 8 8 8

s ig n ifican ce * * »

+ exjDlained at bottom of Tab le 1

* * * p < .001

* * p < .01

* p < .05

n .s . not s ig n if ic a n t.

. 7 3 .

TABLE [8 : P ro po rtio n o f P ap illom a Types and N um ber on A ffec ted Teats

P a ram e te r Value ± SD

Frcmd 26.S>± 8.

% F la t and round 2 0 ,4± 5.9%

% R ice g ra in 5 2 .7± 12,4%

N um ber o f le s io ns 2, 427

N o. o f a ffected teats 472

N um ber p a p illo m a p e r a ffected teat 5.1 ± 1 .0

TABLE 19: N um ber o f P ap illom as of each type o c c u rr in g in th ree m u lt ip l ic i tyg roup ings fro m a se lected* popu la tion .

M u lt ip l ic i ty + 1 - 10 11 - 20 >20 T o ta l

R ice G ra in 83 190 164 437

F rond 35 69 85 189

F la t and Round 37 14 0 51

* tea ts w ith > 10 p ap illom as of one type

+ num be r o f p a p illo m a s p e r teat

, 74,

The d is tr ib u t io n o f p ap illom as acco rd ing to va rio u s m u lt ip l ic i ty

ca tego ries is shown in f ig u re 1. When pap illom as of any type o ccu r in

groups of le ss than. t;en p e r tea t, the d is tr ib u t io n of the F type p a ra lle ls

that of the FR tyjge in both num ber and shape o f cu rve . When p ap illom as

o f any one type occu r in g roups o f > 10, F and FR d is tr ib u tio n d if fe r m a rke d ly —

the F ty)ie o ccu rs in g re a te r frequency w h ile the FR type continues to dec line .

T h is d iffe re n ce is fu r th e r il lu s tra te d in ta b le 19. When teats w ith

m u lt ip l ic it ie s o f > 10 p ap illom as of any one type a re exam ined the F , FR and

RG types occu r in frequenc ies com parab le w ith those fo r the su rvey as a

w hole (fable 18) in the 1-10 p ap illom a p e r tea t g roup . In the 11 - 20

pap illom a p e r tea t g roup, the FR type is reduced and in the > 20 p a p illo m a

p e r teat, g roup , i t has d isappeared a ltog e th e r w h ile the F type has su ccess ive ly

In c re a s e d ,

C o n tra s tin g W il l i the d is tr ib u tio n o f the FR 4- F types, the RG p a p illo m a

type shows a d iffe ren t, d is tr ib u tio n (ftgu re 1) both in num be r and shape of

cu rve a t m u lt ip l ic it ie s o f < 10 p e r tea t. H ow ever, a t m u lt ip l ic it ie s o f > 10

(fable 4) the RG type inc reases in a s im i la r m anner to the F type p a p illo m a .

These in te ra c tio n s a re fu r th e r a m p lif ie d by tab le 20 w h ich shows the

d is tr ib u t io n of the th ree pap illom a types and w he the r they o ccu r as one type

o r in va rio u s com b ina tio ns . T lire e th e o re tic a l m odels w e re tested using

p reva lence f ig u re s lis te d in tab le 20. Two m ode ls , tha t the type d iffe re n ce

was due to one o r th ree separate v iru s /h o s t in te ra c tio n s y ie lded expected

d is tr ib u t io n s at g re a t va ria nce to those observed . The o th e r m ode l, tha t

the type d iffe re n c e was due to two separate v iru s /h o s t in te ra c tio n s , one

c o m p ris in g F T FR type and the o the r RG type y ie lded expected d is tr ib u tio n s

(in b ra cke ts on ta b le 20) no t s ig n if ic a n tly d iffe re n t to those obse rved . The

confounding in fluences o f im m un o lo g ica l re s is ta nce o r s u s c e p tib ility to the

disease led to some d iffe re n ce s between the expected and observed

d is tr ib u tio n s of the to ta l and selected popu la tion B. W here these in fluences

w e re absent, c lose agreem ent between expected and observed fig u re s occu r

(population A ), (The d iffe re n ce between to ta l a ffected tea t num ber in

fa b le s 16 and 18 re s u lts fro m the a cc iden ta l loss of tea ts fro m an un selected

p ro p o rtio n o f c a ttle s laughtered d u r in g p a r t o f one day).

. 75 .

I/)ro

CL

o

E™

.> X

C 3

F ig u re 1: D is fr lb u fio n o f p ap illom as acco rd ing to m u lt ip l ic i ty p e r teat.

. 76 .

T A B LE 20: The d is tr ib u t io n of th ree types o f bovine tea t p a p illo m a o c c u rr in gas one type o r in va rio u s com b ina tions.

P ap illom a Type T and C om bination

T o ta l Survey Selected Population"*

RG 55FR 55F 106F R /F 23

R G /F R 89R G /F 77R G /F R /F 67

50.6

49 .4

T eats % P ap illom as % P ap illom as % P ap illom as %

201112187

79

23 .9(22 . 6)

(77 .4)

350

220

8 .4(8. 2)

166112165

79

29.8(27.9)

72.1 )

T o ta lf 472 100 2427 100 677 100 1,749

+ RG - r ic e g ra in

* se lected population

Note - f ig u re s in b ra cke ts -

FR - f la t and round F - frond

A - teats w ith > 10 p a p illo m a s of any one type B - teats w ith < 10 p a p illo m a s o f any type

expected percentages ca lcu la ted using the. two v iru s /h o s t in te ra c tio n hypo thes is w here F = FR and RG act independently .

Tab le 21 l is ts I he re s u lt o f p a r t ic le m easurem ent o f v iru s fro m p u r if ie d

p re p a ra tio n s o f s ing le case and pooled p ap illom as classed a cco rd in g to th e ir

o r ig in . A ppend ix B conta ins the de ta iled p a r t ic le m easurem ent re s u lts .

E x tra c ts labe lled A a re co ncu rren t p re p a ra tio n s , as a re those labe lled B and

C . T h is means tha t they w ere co llec ted on the same date, hom ogenised and

p u r if ie d s im u lta ne o us ly and exam ined under the e le c tro n m ic rosco p e on the

same day. E x tra c ts RGB, RGC, cB, EC and F R a re pooled sam ples,

w h ile a l l the o the rs have s ing le case o r ig in s . BPV fro m RG s ing le case o r

pooled e x tra c t had a mean p a r t ic le d ia m e te r of 47± 3 m p when RGC is

excluded (see Section 3) BPV fro m TF s ing le case o r pooled e x tra c ts had a

mean d ia m e te r o f 57± 3 m p, w h ile BPV fro m sing le case e x tra c ts of

cutaneous bovine pap illom n (CBPV) had a mean d ia m e te r o f 5 3 .5± 4 .5 m p.

V iru s fro m a dog pap illom a e x tra c t had a mean d ia m e te r o f 65± 2 .4 m p,Using

,77.

the s tudents ' t - te s t, d iffe re n ce s between these groups w ere h ig h ly

s ig n ific a n t (P < .001), how ever w ith in group d iffe re n ce s w ere no t. Two

anom a lies o ccu rre d and a re rep o rte d on table 6. F ir s t ly , one sam ple of a

pooled RG p re p a ra tio n (RG C) had a mean p a r tic le d ia m e te r of 59± 2 mp,

s ig n if ic a n tly d iffe re n t fro m (P C .O l) but s im ila r to the mean d ia m e te rs of

F v iru s d ia in e ie rs . Secondly the FR v iru s mean d ia m e te r of 53± 4 m p was

not s ig n if ic a n tly d iffe re n t from that of CBPV e x tra c ts . These d iffe re n ces

w i l l be d iscussed I'u rthe r in p a r t 3 follow ing&RG-G and FR produced, on

tra n s m is s io n , f ib ro p a p illo m a s , I t is lik e ly that pooled sam ples RGC and

FR contained s ig n ific a n t contam inant f ib ro p a p illo m a v iru s as a re s u lt o f

m acroscop ic c la s s if ic a tio n e r ro rs and p a r tic le d ia m e te r m easurem ents

support th is p ro p o rt io n . F ig u re s 5, 6, 7, 8 and 9 show e le c tron -

m ic ro g ra p h s o f sk in , F la t and Round, R ice G ra in , F rond and Dog Skin

pap illom a v iru s e x tra c ts .

When tw enty individual pap illom as fro m each of the types w e re scraped

and subjected to th ree fre e z e /th a w cyc les , v iru s could be seen in nega tive ly

stained p re p a ra tio n s o f a ll RG and F types exam ined. H ow ever, on ly 13/20

of the FR types y ie lded v is ib le v iru s w ith a fu r th e r two o f the negative FR

p ap illom as y ie ld in g v is ib le v iru s a fte r a repeat sc rap ing around the junc tion

o f n o rm a l and pap illom a to us e p ith e liu m . The re m a in in g fiv e FR les ions

did not y ie ld v is ib le BPV.

The p ro te in com pos itions of v a rio u s p u r if ie d p ap illom a v iru s

p re p a ra tio n s a re shown in fig u re s 10 and 11, The apparent m o le cu la r w e ight

(M j-) o f the m a jo r p ro te in band, w h ich would appear to co rrespond to VP2 o f

human p ap illom a v iru s (G issm an et a l . , 1976), va rie d between Iso la te s ,

T h is band seemed to show a co n s is te n tly h ighe r m o le c u la r w e igh t in teat BPV

iso la tes than in cutaneous BPV. The com para tive mean m o le cu la r w e ights

would be 63, 000 and 56, 000 re s p e c tiv e ly . F ive tea t BPV iso la tes and one

cutaneous BPV iso la te are com pared in fig u re s 10 and 11, T h is has been

extended to ano the r two teat BPV iso la tes and th ree m ore cutaneous BPV

iso la tes (data not shown) w ith the same conc lus ion . F ig u re 10 a lso showed

q ua n tita tive d iffe re n ce s in low m o le cu la r w e ight species w h ich had s im i la r

M^. va lues in a l l BPV iso la te s . The cutaneous BPV p re p a ra tio n showed much

less o f these components than the o the r v iru s p re p a ra tio n s . The canine

. 76.

Figure 5: Electron micrograph of negatively stained preparation of bovine

skin fibropapilloma virus ( C B r V l A ) . XS5,7bO ^ 1 3 2 2 .

. 79.


teat x i b r O j i a p i l l o L i a v i r t i o U'hO • 8 5 7 6 0 ^ 1 5 2 2 .

. 80 .

Figure 7: IHectron micrograph of negatively stained preparation of bovine

teat rice grain papilloma virus t EGA) . X 8 5 7 6 0 1 1 3 2 2 .

. 8 1 .


teat papilloma virus t ?A) . x 8 5 7 6 0 1 1 3 2 2 .

. 8 2 .

m

Figure 9: Flectroii micrograph of negatively stained preparation of dog

skin papilloma virus k D P ) . X 8 5 7 6 0 i 1 5 h 2 .

. 83 .

F ig u re 10: SDS - p o lya c ry la m id e gel e le c tro p h o re s is of bovine pap illom a

e x tra c ts fro m d iffe re n t les ion ty p e s .OariiplGS wore separated on an 11/; acrylamide gel. Lane a; standard marker. protoinsvpnosphorylase-a,94000r;i|. . ; catalase,oOQOO ivi * alcohol dehydrogenase,41000mf <• ; carbonic ' anhydra'c,29000ivit -o ; cytochroine-c,11700M/f .) Lane b)C LxVIa ;lane c) FR plane d;PA;lane e; DP;lane f; EGA,

. 8 4 .

VS

F ig u re 11 : SDS - p o ly a c ry la m id e gel e le c tro p h o re s is o f bovine p ap illom a

e x tra c ts fro m d if fe re n t lesion types, b ample3 wore separated on an 8y, acrylamide gel. a) standard proteins B - g a l a c t o s e , l;30000iv:f rest as for f i g a i r c l i ) with the e x c e p t i o n of cytochroine-c; ; 6 ; c b , V i u ; c )C B P V ln ; d ) F B ; e)RdB; f ) C B r V 2 . •

. 8 5 *

TA B LE 21 :

E x t ra c t of

Mean BPV p a r t ic le d ia m e te r measured on e le c tronm ic rog raphs®

o f nega tive ly stained p u r i f ie d e x trac ts o f va r io us pap il lom a

s a m p le s .

Mean D ia m e te r ± SD (mp) N um ber Measured

RG A 47.6 ± 2 . 9 25

RG B 46.7 ± 3 .5 200

RG C 58.7 ± 2 .2 100

F A 5 7 .6 ± 2 .6 50

F B 56.5 ± 3 . 3 125

F C 56 .9 ± 3 .4 75

FR 5 3 .0 ± 4.1 175

+CBPV1, A 5 1 .7 ± 4 .8 25

CBPVl B 53.6 ± 4 .5 75

CBPV2 5 4 .9 ± 3 .6 150

X DP 65.1 ± 2 .4 50

+ cutaneous bovine pap illom a v i ru s ex trac t

X dog pap il lom a e x tra c t

explained at foot o f table 20 «

0 ac tua l m agn if ica t ion x 8i§ 760 ± 1322.

8 b o

pap il lom a v i r u s showed a d is t in c t iv e pa tte rn , w ith two bands in the "VP 2"

reg ion wh ich did not com lg ra te w ith any band in the BPV p re p a ra t io n s .

T h e re was a lso a v e ry p ro m in en t band at 15, 000 M’ „ Some

p re p a ra t io n s (F ig . 11, lanes b and c) showed an excess of high m o le c u la r

we ight bands ove r any band in the expected "VP 2" re g io n . Since the

p u r i f ic a t io n p rocedu re involved only a s ing le CSCl g ra d ie n t step, i t seems

l ik e ly that these bands rep resen t t issue p ro te in con tam ina tion . Although

these p re pa ra t io ns contained v i ru s when examined by e lec tron m ic roscopy ,

la rg e r quan ti t ies of t issue had been extrac ted inc re as ing the like lihood of

seeing m in o r contam inant species in SDS.

2 ,4 D iscuss ion

N o rm a l ly bovine cutaneous f ib ro pa p i l lo m a a ffec t cattle less than two

years of age, has a w e l l recognised v i r a l ae t io logy and is a se lf l im i t in g

d isease. (F renz , 1941; Bagdonas and Olson, 1953). The re su lts of a

recen t su rvey suggest that bovine a l im e n ta ry p a p i l lom a to s is p resen ts a

d i f fe re n t ep ide m io lo g ica l p ic tu re w ith an o ve ra l l p reva lence of 19%. No

s ig n if ican t d if fe ren ces w ere found between catt le aged < 3 years and ca tt le

aged > 3 years in e i th e r pap il lom a preva lence o r m u l t ip l i c i t y . V i r u s - l i k e

p a r t ic le s w e re found in fo u r of seven pap il lom as examined as t l i in sections

under the EM w h ile l ig h t m ic rosco p y o f 100 cases revealed a 13% prevalence

of type A. in t ra n u c le a r inc lus ion bodies (Ja rre tt et a l . , 1978a).

T h is p a r t re p o r ts that teat pap i l lom a to s is p resen ts an ep idem io log ica l

s itua tion m a rk e d ly d i f fe re n t to that reported p re v io u s ly fo r e i th e r cutaneous

o r a l im e n ta ry p a p i l lo m a to s is . W ith the exception of one cow, a l l pap il lom a

observed w ere l im i te d to the h a ir le s s po r t ion of the teats w ith random

d is t r ib u t io n ove r that l im ite d a re a . The one exception was a cow showing

a m ixed type population of 155 teat pap il lom a on the f ive teats she possessed-

two of these pap il lom a w ere 3 cm above the h a i r - l in e , the re s t w e re re s t r ic te d

to the h a i r le s s port ion of tlie tea ts . C oncurren t exam ination of the re s t of

the in tegument of each of the 72] ca tt le y ie lded two an im a ls w ith cutaneous

f ib ro -p a p i l lo m a in s is and four w ith sc ro ta l pap il lom as , a respec t ive

p reva lence o f 0.3% and 2.4%, W li i le pap il lom as were never observed w ith in ,

the teat canal, they frequently fo rm ed c lu s te rs around the teat o r i f ic e , '

. 6 7 ,

m aking it qu ite conceivable that m i lk f ro m these a n im a ls would contain v i ru s

re leased by the s u p e r f ic ia l traum a of suck ling ca lves o r machine m i lk in g .

The e ffect of p as teu r isa t ion on in fe c t iv i ty of BPV ex trac ted f ro m the th ree

types of les ions w i l l be reported in p a r t 6 of th is the s is .

W h ile the re w e re no s ign if ican t d if fe rences between h e i fe rs (nu ll ipa rous

fem a les ) and cows (porous fem ales) in the p ro p o r t io n a l num be r o f an im als

affected, there was a s ign if ican t increase in the num ber o f a ffected teats per

a n im a l . Male an im a ls showed a s ig n if ica n t ly low er p reva lence of teat

p a p i l lo m a to s is w ith few er teats a ffected p e r an im a l. The m a le - fe m a le

d i f fe rences m ay s im p ly be a re f le c t io n of re la t iv e d if fe re n ces in the size of

tea ts , but ho rm ona l o r behav ioura l d i f fe rences cannot be d is re ga rd e d . The

c o w -h e i fe r d if fe rences ind icate a h ighe r preva lence w ith in c re as ing age o r

p a r i ty ,b u t a lso that teat p ap i l lom a to s is is w idespread in the popula tion before

f i r s t ca lv ing .

Separation of the th ree pap illom a types on m ac ro sco p ic appearance

proved re la t iv e ly easy. There w ere cases where ind iv idua l les ions w ere

classed as FR when la te r deta iled exam ination proved them to be e ith e r

F o r RG, but, re la t iv e to the to ta l num bers invo lved, the system worked w e l l ,

The u n l ike ly f ind ing that the to ta l num ber of p ap i l lom as com prised

a pp ro x im a te ly h a lf (52,7%)RG types and h a l f F + FR (26.9% and 20.4%

re s p e c t iv e ly ) coupled w ith the fact that F and FR both possessed the common

fa c to r of connective t issue invo lvem ent, led to the f i r s t suspic ion that teat

p a p i l lo m a to s is was a mixture of two c lose ly re la ted but separab le disease

p ro cesse s . T h is suspic ion was enhanced by the f ind ing ( f ig . 1) that F and

FR had s im i la r d is t r ib u t io n s at lo w e r m u l t ip l ic i t ie s (<10) w h i le RG had a

d is t r ib u t io n d i f fe re n t in both num ber and shape of cu rve . The d b t r ibutions

a t h igh (>10) m u l t ip l ic i t ie s , presented a d i f fe re n t p ic tu re where RGand F

increased ra p id ly w h ile FR continued i t s ’ decline (table 19). T h is la t te r

fea tu re suggested that FR is a re g re s s in g fo rm of F , exp la in ing the e a r l ie r

observa tion that, o v e ra l l , F exceeds FR in num ber (26.9% compared w ith

20.4%).

When three hypotheses w e re put, that these type d if fe rences w e re due

to 1, 2 o r 3 separate v i ru s /h o s t in te rac t ion s , the on ly hypothesis wh ich

y ie lded com parab le expected and observed p ro p o r t io n s in les ion d is t r ib u t io n .

8 8

(table 20) was that two v i ru s /h o s t in te rac t ions were o c c u r r in g . I f one, F and

FR and the o t l ie r , RG a re treated as separate, independent p rocesses then

they would be expected to occu r together in 77,4% of the to ta l pap il lom a

popu la tion , 72.1% of the selected population 13 (<10 ) and 91.8% of

the selected popula tion A (> 10 ). Converse ly , they would occur alone

in 22.4% of the to ta l, 29.9% of selected B and 8.2% o f se lected A pap il lom a

popu la tions. The respec t ive observed percentages a re 23 .9% , 29.8% and

8.4% and these a rc not s ig n if ic a n t ly d i f fe re n t f ro m the expected calculated

using the two fa c to r hypo thes is . Observed and expected popula tions were

a t g rea t va r iance when a s ingle o r th ree fa c to r hypothesis w e re used.

A g reem en t between expected and observed f igu res is best in selected

population A w ith only a d if fe rence of 0,2% , H igh m u l t ip l ic i t y popula tion A

a lso coal a ins a low FR complem ent so i t is l ik e ly that th is population is

im m u n o lo g ic a l ly less competent to deal w ith the d isease . In the to ta l

su rvey and population B, the confounding in fluences o f ind iv id ua l im m u n ity

and s u s c e p t ib i l i ty m ust lead to g re a te r va r iance between expected and

observed frec juenc ies.

The dem onstra tion of pap il lom a v i ru s in sc rap ings f ro m a l l th ree types

rep resen ts a new f ind ing . The technique used has not been p re v io u s ly

repor ted and may have app lica t ion in c l in ic a l m ed ic ine both because i t a l low s

sam p ling of in d iv idua l p ap il lom as and because i t y ie lds an answ er w ith in an

hour o f o r ig in a l sam p ling .

F u r th e r support fo r the suggestion that FR is a re g re s s in g fo rm comes

f ro m the f ind ing that v i ru s was m o re d i f f ic u l t to obtain by scrape sam pling

(in 25%, v i r u s could not be dem onstra ted ).

M easurem ent of BPV p a r t ic le d iam e te rs on e lec tron m ic ro g ra p h s o f

nega tive ly stained p re pa ra t ions of CsCl p u r i f ie d e x tra c ts proved d i f f i c u l t and

t im e consum ing. A l l F samples showed a s ig n if ic a n t ly h ighe r mean

d ia m e te r than CBPV sa up Res w h ile two of the three RG samples were

s ig n i f ic a n t ly lo w e r . The mean p a r t ic le d iam ete rs re po r ted in th is paper a re

on average 5 m p h ig l ic r than those reported p re v io u s ly - th is may be due to the

fac t that nega tive ly stained p repa ra t ions were used o r that our technique is

d i f fe re n t to that used in o the r la b o ra to r ie s .

89

The cutaneous and teat BPV p repa ra t ions wh ich have been examined

showed d is t in c t po lypeptide com pos it ions . Th is d if fe rence seems to be

re p ro d u c ib le . To date seven teat BPV and four cutaneous BPV iso la tes have

been exam ined. G issman et a l . , (1976), showed that one human pap il lom a

v i r u s (I-IPV) iso la te , MPV 4, was unre la ted to iso la tes HPV 1-3, and showed

a d i f fe re n t po lypeptide d is t r ib u t io n . T liey w e re also unre la ted by DNA

endonuclease cleavage pa tte rn , CRNA hyb r id isa t io n and s e ro lo g ic a l

s p e c i f ic i ty . No such c r i t e r ia a re ye t ava i lab le fo r BPV iso la tes , but i t w i l l

be in fo rm n t iv e to com pare them in th is m anner. Some ch a ra c te r isa t io n of

HPV p ro te in s has been reported by P f is te r et a l . , (1977) who found VP2, 3

and 4 to be h ig l i ly re la ted by exam in ing BrCN cleavage p a t te rn s . They w ere ,

however, unable to es tab lish that VP3 was a p ro te o ly t ic cleavage p roduc t of

VP 2 by th is technicjue, and suggested that the d if fe rences may be due

to acé ty la t ion , deam ination o r g ly c o s y la t io n . T l i is is of p a r t i c u la r in te res t

w ith regard to the teat " r i c e - g r a in " v i r u s which g e n e ra l ly showed a second

m o le c u la r species, equ im o la r w ith "VP 2" and of s l ig h t ly lo w e r m o le cu la r

w e igh t.

Q uan tita t ive d if fe re nces in low m o le cu la r we ight species (12 to

15, 000 M y) w e re a lso found. Cutaneous BPV showed v e ry l i t t l e of these

components. A p rob le m which was encountered w ith some v i r u s p repa ra t io ns

was the presence o f what appear to be contaminant t issue p ro te in s . The

v i r u s p u r i f ic a t io n p ro to co l wh ich was used was as b r ie f as poss ib le , since

i t was thought im p o r ta n t to leave the v i r u s s t ru c tu re in tac t .

2 .5 S um m ary

In a su rvey o f 721 ca tt le passing through Glasgow a b a t to ir , 36% had

teat p a p i l lo m a to s is . A ffec ted a n im a ls possessed an average of f ive

p ap i l lom as on each of two affected teats. S ign if ican t sex d if fe rences o ccurred

in the p reva lence of the d isease, and n u l l ipa rous fem ale ca tt le had s ig n if ic a n t ly

few er teats affected than parous fem a les .

When ind iv idua l pap il lom a were d iv ided accord ing to m acroscop ic

appearance, 27% w ere of the frond(F) type; 20% w e re o f the f la t and round

(FR) type and 53% w e re r ic e g ra in (RG) les ions . The F and FR types had the.

common fea tu re of connective t issue invo lvem ent and showed s im i la r

. 90 .

d is t r ib u t io n s in the popula tion exam ined. The RG type had a s ig n if ic a n t ly

d i f fe re n t d is t r ib u t io n and lacked connective t issue invo lvem ent. The

observed and expected frequencies of association between the t l i ree t^rpes

w e re not d i f fe re n t only when a model assum ing two separate, independent

v i r u s /h o s t in te rac t ion s was used. In th is model F and FR types formed,

one and RG the o the r v i ru s /h o s t in te ra c t io n . F ro m h ighe r m u l t ip l ic i t y

cases, i t was concluded I hat FR could be a re g re ss in g fo rm o f the F type.

Bovine Pap il lom a V iru s (BPV) could be dem onstra ted in a l l F and RG

types exam ined, but only in 75% of FR type p ap i l lo m a s . Mean p a r t ic le

d ia m e te rs of RG, F^ cutaneous f ib ro p a p i l lo m a and canine skin pap il lom a

v iru s e s w e re s ig n i f ic a n t ly d i f fe re n t f ro m each o th e r .

Separate p u r i f ie d v i r a l e x tra c ts f ro m 4 cutaneous and 7 teat pap il lom a

samples w e re compared by p o ly a c ry la m id e gel e le c tro p h o re s is . A l l

p u r i f ie d teat v i r u s samples showed a m a jo r capsid p ro te in b^-tid w h ich had a

cons is ten t ly h ighe r apparent m o le c u la r we ight than com parab le cutaneous

v i r u s e x tra c ts .

T h is p a r t suggests that two d i f fe re n t BPV v i r u s /h o s t in te rac t io n s occu r

in bovine teat p a p i l lo m a to s is . V i ru s ex trac ted f ro m teat p ap il lom a was .

s ig n i f ic a n t ly d i f fe re n t f ro m the v i r u s of cutaneous f ib ro p a p i l lo m a to s is in

both p a r t ic le d ia m e te r and apparent m o le c u la r we ight o f m a jo r v i r a l capsid

p ro te in .

, 91 ,

C I-IA PT ER 3

IN V IV O E F F E C T S O F B O V IN E P A P I L L O M A V IR U S :

E X P E R I M E N T A L T R A N S M IS S IO N TO C A T T L E O F B O V IN E

P A P I L L O M A V IR U S (B P V ) E X T R A C T E D F R O M

M O R P H O L O G I C A L L Y S E P A R A B L E T E A T A N D C U T A N E O U S

L E S IO N S A N D T H E E F F E C T S O F I N O C U L A T IO N O F B P V

T R A N S F O R M E D F O E T A L B O V IN E C E L L S .

3 .1 I n t r o d u c t i o n

B o v in e p a p i l l o m a v i r u s (B P V ) has been e x t r a c t e d f r o m a

v a r i e t y o f le s io n s o c c u r r i n g on d i f f e r e n t a n a to m ic a l s i te s in c a t t le .

B P V w as fo u n d to be the c a u s a t iv e a g e n t o f c u ta n e o u s

f i b r o p a p i l l o m a s by C r e e c h ( 1 929), g e n i ta l f i b r o p a p i l l o m a s

(M c E n te e , 1952), and i t m a y be a s s o c ia te d w i t h c h r o n ic e n z o o t ic

h a e r n a tu r ia o f c a t t le (O ls o n e t a l . , 1965). A t y p i c a l c u ta n e o u s

p a p i l l o m a s have been show n to c o n ta in B P V ( B a r t h o ld e t a l . , 1974).

B P V has been e x t r a c te d f r o m a l im e n t a r y p a p i l l o m a s ( J a r r e t t e t a l . ,

1978); te a t p a p i l l o m a s , te a t f i b r o p a p i l l o m a s and te a t r i c e g r a in

le s io n s , s i m i l a r to f o c a l e p i t h e l ia l h y p e r p la s ia in m a n (C h a p te r 2).

In v i t r o B P V t r a n s f o r m s c u l t u r e s o f f o e ta l b o v in e s k in ,

c o n ju n c t iv a , m e n in g e s a nd p a la te (C h a p te r 7 ). T h is chapter r e p o r t s

the r e s u l t s o f e x p e r im e n t a l i n o c u la t io n o f c a lv e s w i t h d i f f e r e n t

B P V e x t r a c t s , B P V t r a n s f o r m e d fo e ta l b o v in e c e l l s , and

s u b s e q u e n t c h a l le n g e w i t h the sam e B P V e x t r a c t s .

3 . 2 M a t e r i a l s and M e th o d s

3 . 2 . 1 L e s io n s

B P V e x t r a c t s w e re d e r iv e d f r o m f i v e c a t t le , each

a f fe c te d w i t h o n ly one o f the f o l lo w in g m o r p h o lo g i c a l l y

s e p a ra b le le s io n ty p e s ,

A . R ic e g r a in le s io n s o f the b o v in e te a t (C h a p te r 2),

h i s t o l o g i c a l l y s i m i l a r to f o c a l e p i t h e l i a l h y p e r p la s ia

in m a n ( P r a e t o r i n s -C la u s e n , 1972).

92 .

B . P a p i l l o m a w i t h o u t f i b r o m a , p r e v io u s l y d e s c r ib e d

as a t y p ic a l w a r t s on b o v in e s k in ( B a r t h o ld e t a l . ,

I 9 7 4 ) and a ls o r e p o r t e d on the b o v in e te a t

( C h a p te r 2).

C . F i b r o p a p i l l o m a o c c u r r i n g on the b o v in e tea t ,

p r e v io u s l y d e s c r ib e d as f l a t and ro u n d le s io n s

(C h a p te r 2).

D . T y p ic a l f i b r o p a p i l l o m a as d e s c r ib e d in the

l i t e r a t u r e o c c u r r i n g on a n t e r i o v e n t r a l s k in s i te s

(B a g d o n a s and O ls o n , 1953).

E . F i b r o p a p i l l o m a , p r e s e n t in g as c a u l i f l o w e r l i k e

le s io n s a f fe c t in g the a n o - g e n i t a l r e g io n s a n d the

v e n t r a l a b d o m in a l w a l l (M c E n te e , 1952; T w e d d le

and W h i te , 1977).

L e s io n s w e re r e m o v e d a n d s to r e d s e p a r a te ly in

P B S / g l y c e r o l a t 4 ° C . One le s io n f r o m e a ch o f the f iv e

ca s e s w a s s a m p le d and p la c e d in 10% f o r m o l s a l in e f o r

f u r t h e r h is t o p a th o lo g i c a l e x a m in a t io n .

3 . 2 . 2 V i r u s E x t r a c t i o n

A f t e r h i s t o l o g i c a l c o n f i r m a t i o n o f l e s io n ty p e , the

s to r e d s a m p le s w e r e h o m o g e n is e d u s in g a S i l v e r son b le n d e r

in to a 10% w / v c ru d e s u s p e n s io n in p ho s p h a te b u f fe r e d

s a l in e (P B S ) . T h is s u s p e n s io n w a s c l a r i f i e d u s in g an

S W 27 r o t o r a t 1 0 ,0 0 0 r . p . m . f o r 20 m in u te s i n a B e c k m a n

L -2 50 u l t r a c e n t r i f u g e . The r e s u l t i n g s u p e r n a ta n t w as

the n la b e l le d and s to r e d a t - 7 0 ° C u n t i l f u r t h e r u se .

S u s p e n s io n s w e r e n e g a t i v e ly s ta in e d w i t h p h o s p h o tu n g s t ic

a c id ( P T A ) a nd e x a m in e d u n d e r the e le c t r o n m ic r o s c o p e

(E M ) to c o n f i r m the p re s e n c e o f B P V .

3 . 2 . 3 F o e ta l B o v in e C e l ls

T r a n s f o r m a t i o n s e n s i t i v e fo e ta l b o v in e m e n in g e a l

■ 93 ,

c e l l s ( F B M C ) and f o e t a l b o v in e s k in c e l l s (F B S C ) w e r e

p r e p a r e d as e ls e w h e re d e s c r ib e d (C h a p te r 7). A

t r a n s f o r m e d c u l t u r e w a s p r e p a r e d by in f e c t in g F B S C and _ 2

F B M C w i t h a 10 d i l u t i o n o f a 10% w / v B P V e x t r a c t o f

le s io n s c la s s e d as D a b o ve . P a r a l l e l u n t r a n s f o r m e d and

t r a n s f o r m e d F B S C and F B M C w e r e p a s s a g e d a t w e e k ly

i n t e r v a l s u s in g B a s a l E a g le s M e d iu m ( B M E ) w i t h 10%

added f o e ta l b o v in e s e r u m (F B S ) . C u l t u r e s f r o m p assages

1 0 - 1 2 w e r e u sed f o r a n im a l i n o c u la t io n . U s e d c u l t u r e

m e d iu m w a s c o l le c te d , p r e c ip i t a t e d a t 4 ^C w i t h 50% added

s a tu r a te d a m m o n iu m s u lp h a te , re s u s p e n d e d in PBS and

s u b je c te d to d i f f e r e n t i a l c e n t r i f u g a t io n b e fo re C s C l

g r a d ie n t p u r i f i c a t i o n and E M e x a m in a t io n f o r the p re s e n c e

o f B P V . T r a n s f o r m e d a n d c o n t r o l c u l t u r e s w e r e g ro w n on

5 c m p la s t i c p e t r i d is h e s f o r th re e d ays , the m e d iu m w a s

r e m o v e d and c e l l s w e r e f i x e d in s i tu u s in g g lu ta r a ld e h y d e .

A f t e r f i x a t i o n , the c e l l s w e r e re m o v e d u s in g a r u b b e r

p o l ic e m a n and f u r t h e r p ro c e s s e d f o r th in s e c t io n E . M .

3 . 2 . 4 A n i m a l I n o c u la t io n

T e n c a lv e s 2 - 4 m o n th s o ld w e re ra n d o n a ly

d iv id e d in to t h r e e g ro u p s . (1) F iv e c a lv e s w e r e in o c u la te d

w i t h v i r u s i s o la te s f r o m le s io n s A - E . (2) T h r e e c a lv e s

w e r e in o c u la te d w i t h t r a n s f o r m e d c e l l c u l t u r e s . (3) T w o

c a lv e s w e r e in o c u la te d w i t h c o n t r o l c e l l c u l t u r e s . T w o

c a lv e s , one in o c u la te d w i t h le s io n D v i r u s and one

in o c u la te d w i t h le s io n D B P V t r a n s f o r m e d c e l l s (F B S C )

w e r e k e p t is o la te d f r o m the o th e r c a lv e s and f r o m each

o th e r . The o th e r c a lv e s w e r e k e p t s e p a r a te ly i n tw o g ro u p s

o f f o u r - the v i r u s in o c u la te d a nd the c e l l in o c u la te d -

e x c e p t d u r in g the c o n ta c t s e c t io n o f the e x p e r im e n t . V i r u s

in o c u la te d c a lv e s w e r e g iv e n 10% w / v s u s p e n s io n s o f B P V

f r o m le s io n s A - E bo th as 10 i n t r a d e r m a l in je c t io n s a n d b y

s c a r i f i c a t i o n on b o th s id e s o f the to r s o , each c a l f r e c e iv in g

. 9 4 .

v i r u s f r o m o n ly one le s io n ty p e . C e l l in o c u la te d c a lv e s

r e c e iv e d te n i n t r a d e r m a l in je c t io n s o v e r the lu m b a r a re a

o f the t o r s o as w e l l as th re e s u b l in g u a l ly . E a c h in je c t io n

c o n ta in e d 10 c e l l s in 0 .2 m l PBS, o r 0 . 2 m l o f the 10% w / v

B P V s u s p e n s io n . A l l in o c u la c o n ta in e d 10 m g / m l o f

s t e r i l i s e d , f i l t e r e d , a c t i v a te d c h a r c o a l to a c t as a m a r k e r

f o r b io p s y s a m p l in g .

3 . 2 . 5 B io p s y S a m p le s

A n i m a l s w e r e s a m p le d a t f o r t n i g h t l y i n t e r v a l s a t

b o th the i n t r a d e r m a l and s c a r i f i c a t i o n s i te s th r o u g h o u t the

p e r io d o f the e x p e r im e n t . E a c h b io p s y s a m p le w a s d iv id e d

in to tw o , one p a r t w a s f i x e d in 10% f o r m o l s a l in e , the o th e r

w as f r o z e n a t -7 0 ° C f o r f u t u r e u se . L e s io n s w e r e s c r a p e -

s a m p le d as d e s c r ib e d e a r l i e r (C h a p te r 2) and e x a m in e d

u n d e r the E M to d e te r m in e the p re s e n c e o f B P V . A s

in d ic a te d on"T ab le% , le s io n s f r o m the s c a r i f i c a t i o n s i te s

w e r e h a r v e s te d a t i n t e r v a l s and s to r e d in 50% P B S / G ly c e r o l

f o r v i r u s e x t r a c t i o n . B lo o d w a s c o l le c te d a t f o r t n i g h t l y

i n t e r v a l s f o r h a e m a to lo g ic a l e x a m in a t io n a n d s e r u m w as

s to r e d a t - 2 0 ° C u n t i l r e q u i r e d f o r f u r t h e r use (see C h a p te r 8)

3 . 2 . 6 C h a l le n g e E x p e r im e n t

A n i m a ls w e r e c h a l le n g e d by tw o m e th o d s . The

c a lv e s ( e x c lu d in g the tw o is o la te d c a lv e s ) w h ic h r e c e iv e d

v i r u s a nd show ed v i s ib le le s io n s w e re p la c e d i n c o n ta c t w i t h

the c e l l in o c u la te d c a lv e s f o r a p e r io d o f e ig h t w e e k s . A l l

a n im a ls w e r e e x a m in e d a t w e e k ly i n t e r v a l s a nd the p re s e n c e

a nd type o f any le s io n s d e v e lo p in g a t n o n - in o c u la t io n s i te s

w e r e n o te d . The second m e th o d in v o lv e d the c h a l le n g e o f

e v e r y c a l f w i t h v i r u s f r o m each o f the f i v e le s io n ty p e s .

S c a r i f i c a t i o n s i te s w e r e p r e p a r e d by c l ip p in g the h a i r f r o m

f i v e 10 X 20 c m m a j o r s k in a re a s le a v in g a t le a s t 10 c m o f .

u n d ip p e d h a i r c o v e re d s k in b e tw e e n a d ja c e n t a r e a s .

. 9 5 .

C h a l le n g e s i te s w e r e o b s e rv e d a t w e e k ly i n t e r v a l s and the

p re s e n c e and typ e o f a n y le s io n w as n o te d .

3 . 2 . 7 P o s t M. o r te nr E x a m in a t i on

A l l c a lv e s w e r e s u b je c te d to a d e ta i le d p o s t

m o r t e m e x a m in a t io n tw e lv e w e e k s a f t e r c h a l le n g e by

s c a r i f i c a t i o n . A l l s y s te m s w e r e e x a m in e d m a c r o s c o p ic a l l y

in d e ta i l , w i t h s p e c ia l a t t e n t io n g iv e n to e p i t h e l i a l t is s u e s

in v o lv in g the a l im e n t a r y , r e s p i r a t o r y and g e n i t o - u r i n a r y

t r a c t s . L e s io n s fo u n d w e re f i x e d in 10% f o r m o l s a l in e and

s u b s e q u e n t ly p ro c e s s e d f o r h is t o p a th o lo g i c a l e x a m in a t io n .

3 . 2 . 8 H is to pa th o lo g y

A l l s a m p le s w e r e r o u t i n e l y s ta in e d w i t h

h a e m a to x y l in and e o s in , a p p r o p r ia t e s p e c ia l s ta in s w e r e

used w h e n n e c e s s a r y .

3 . 3 Re s u i ts

3 . 3 . 1 H i s to pî th o lo g y

A ) F ig u r e 12 show s the h is t o l o g i c a l a p p e a ra n c e o f b o th

the le s io n s f r o m w h ic h B P V w a s e x t r a c t e d and those

p ro d u c e d by t r a n s m is s io n to c a l f 68882 ( ta b le 22).

M a c r o s c o p i c a l l y , the r i c e g r a in le s io n s a re w h i te

o r f le s h c o lo u r e d , s m a l l , e le v a te d n o d u le s , le s s

than 1 c m d ia m e t e r w i t h o u t p a p i l l o m a to u s f r o n d s

(a c u m in a te , f i l i f o r m p r o je c t io n s o f e p i t h e l iu m ) .

In s e v e re c a s e s , le s io n s m a y c o a le s c e to c o v e r

la r g e a r e a s . M i c r o s c o p i c a l l y , the le s io n s a re

c h a r a c t e r i s e d by a f o c a l e p i t h e l ia l h y p e r p la s ia w i t h

m a r k e d a c a n th o s is but w i t h o u t p a r a k e r a t o s i s . T h e r e

is l i t t l e o r no h y p e r k e r a t o s i s e x c e p t in a r e a s between

c o n n e c t iv e t i s sue p a p i l l a e . A n in c r e a s e d c e l l u l a r

d e n s i ty o f the e p i t h e l iu m w i t h m a in l y h o r i z o n t a l

th ic k e n in g o f the e p i t h e l ia l r e te p eg s , r e n d e r s the

. 9 6 .

* * » H.VV

»

b

F IG U R E 12 - (a) H is t o lo g i c a l a p p e a ra n c e o f te a t r i c e g r a in

le s io n f r o m w h ic h B P V w a s e x t r a c t e d , (b)

T r a n s m i t t e d r i c e g r a in le s io n on s k in o f c a l f

68882 . T h e s e le s io n s a re s i m i l a r to f o c a l

e p i t h e l i a l h y p e r p la s ia in m a n , a n d a re

n e i t h e r p a p i l l o m a s n o r f i b r o p a p i l l o m a s .

S ta in H & E B a r = I r n m .

97

T A B L E 22 T H E D E V E L O P M E N T ® A N D R E G R E S S IO N ° O FL E S IO N S P R O D U C E D A T B P V IN O C U L A T IO N S IT E S .

C A L F 68882 68886 68888 68891 68893

B P V S O U R C E - A E B D C

T IM E P O S T I N O C U L A T IO N

(w e e k sj2 4 1- 4 4 4

4 44 4 4 4 44

6 141 4H o 41- 41- 44

8 144 1441 ooo 141 441 o 144

10 m 141 oo 1441 3441 ooo 3444

12 441 44 o 414-1 14-1 oo H44

14 414 4 o 11-41 41 o 1441 o

16 141- 1441 4 o 4444 oo

18 114 1141 4434 oo

20 141- 1414 1414 o

22 4 1441 1441

24 4 4444 1-44-1

L E S IO N T Y P E A E B D C

r e g r e s s io n o l le s io n s o - s m a l l a re a s o f ly m p h o c y te a c c u m u la t io n oo = la r g e m a s s e s o f ly m p h o c y te s a c c u m u la t in g . ooo = la r g e m a s s e s o f l y m p h o c y te a c c u m u la t io n w i t h n e c r o s is o f in v o lv e d t i s s u e s .

l e s io n ty p e s A - E d e s c r ib e d in M a t e r i a l s & M e th o d s se c t io n .

d e v e lo p m e n t o f le s io n s + v is ib le s c a r i f i c a t i o n l in e s-14 th ic k e n e d s c a r i f i c a t i o n l in e s H F le s io n s "F I c m d ia m e te r4444 le s io n s > 1 c m d ia m e t e r .

c o n n e c t iv e t is s u e p a p i l la e e x t r e m e l y th in ,

c o n ta in in g o n ly b lo o d and ly m p h v e s s e ls w i t h

a lm o s t no c o n n e c t iv e t is s u e s t r o m a . The s t r a t u m

g r a n u lo s u m is m a r k e d ly th ic k e n e d w i t h c e l l s

s h o w in g a n g u la r b a s o p h i l i c i n t r a c y t o p la s m ic

in c lu s io n s . In the u p p e r s t r a t u m s p in o s u m ,

o c c a s io n a l b a s o p h i l i c i n t r a n u c le a r in c lu s io n s a re

seen . T h e r e is no d e r m a l f i b r o m a a nd the d e r m o -

e p id e r m a l ju n c t i o n is w e l l d e f in e d in e a r l y le s io n s .

On t r a n s m is s io n , le s io n s w e r e f i r s t seen 4 w e e ks

p o s t in o c u la t io n and p e r s is t e d t i l l w e e k 20

( ta b le 22). R e g r e s s io n o f the le s io n s w a s

a c c o m p a n ie d by a c h r o n ic i n f l a m m a t o r y i n f i l t r a t e

s u r r o u n d in g a r e a s o f a d is r u p t e d e p i d e r m o - d e r m a l

f u n c t io n , u l t im a t e l y le a d in g to s lo u g h in g o f

a f fe c te d e p i t h e l iu m w i t h r e p la c e m e n t by p lu g o f

f i b r i n a nd i n f l a m m a t o r y c e l l s . R e g r e s s io n w a s n o t

a c c o m p a n ie d by a m a s s iv e l y m p h o c y te a c c u m u la t io n

as seen w i t h f i b r o p a p i l l o m a s . P r e s e n c e o f r i c e

g r a in le s io n s d id n o t p r e v e n t the t r a n s m is s io n o f

f i b r o p a p i l l o m a s on c a l f 68882 ( ta b le 23). The

r e g r e s s io n o f f i b r o p a p i l l o m a s ( d e s c r ib e d l a t e r ) w a s

n o t a c c o m p a n ie d by the r e g r e s s io n o f r i c e g r a in

le s io n s .

B) F ig u r e 13 show s the h i s t o l o g ic a l a p p e a r a n c e o f b o th

the le s io n s f r o m w h ic h B P V w a s e x t r a c t e d and tho se


M a c r o s c o p ic a l l y , p a p i l l o m a s a r e c i r c u m s c r i b e d ,

e x o p h y t ic g r o w th s w i t h a p a p i l l o m a to u s s u r fa c e

( f r o n d le n g th ^ 2 m m ) w h ic h m a y be s e s s i le o r

p e d u n c u la te d . M i c r o s c o p i c a l l y , th e y a r e

c h a r a c t e r i s e d by a p a p i l lo m a to u s s u r fa c e w i t h

c o n s p ic u o u s h y p e r k e r a t i n i s a t i o n . P r e d o m in a n t l y

h y p e ro r th o jS fe ra t in is a t io n bu t w i t h o c c a s io n a l a r e a s '

o f p a r a k e r a t o s i s . The s t r a t u m g r a n u lo s u m is

. 9 9 ,

#

F IG U R E 13 (a) H is t o lo g i c a l a p p e a ra n c e o f te a t

p a p i l l o m a w i th o u t f i b r o m a f r o m w h ic h

B P V w a s e x t r a c te d (b) t r a n s m i t t e d

le s io n sh o w in g p a p i l l o m a w i t h o u t

f i b r o m a c o m p o n e n t on s k in o f c a l f

68888. T hese le s io n s a r e s i m i l a r to

a ty p ic a l w a r t s p r e v io u s l y r e p o r t e d oh

b ov ine s k in .

S ta in I I & E . B a r = 1 m m .

ion

m a r k e d l y th ic k e n e d , e s p e c ia l l y in the a r e a s b e tw e e n

the p a p i l l o m a to u s e le v a t io n s w h ic h f o r m a r o u n d th in ,

e lo n g a te d c o n n e c t iv e t is s u e p a p i l l a e . E p i t h e l i a l r e te

b r id g e s o f te n show a c h a r a c t e r i s t i c in w a r d b e n d in g

a t the le s io n m a r g in s . The s t r a t u m g r a n u lo s u m

c o n ta in s dense , b a s o p h i l i c i n t r a c y t o p la s m ic

i n c l u s io n s , w i t h c e l l s c o n ta in in g i n t r a n u c le a r

in c lu s io n s in l a t e r le s io n s . T he d e r m o - e p id e r m a l

j u n c t i o n is w e l l d e f in e d in e a r l y le s io n s and th e re

is no a c c o m p a n y in g d e r m a l f i b r o m a . On

t r a n s m is s io n , le s io n s w e re f i r s t n o t ic e d a t 8 w e e k s

p o s t i n o c u la t io n and p e r s is t e d u n t i l p o s t m o r t e m a t

w e e k 25. In b io p s y s a m p le s f r o m w e e k 14 t i l l w e e k

25, a c h r o n ic i n f l a m m a t o r y r e a c t i o n c o u ld be seen

w i t h the o c c a s io n a l i n t e r r u p t i o n o f a f f e c te d e p id e r m o -

d e r m a l j u n c t i o n s . L a r g e a c c u m u la t io n s of

l y m p h o c y te s , a c c o m p a n y in g r e g r e s s io n o f f i b r o -

p a p i l l o m a s (see l a t e r ) w e r e n o t seen . The p re s e n c e

o f p a p i l l o m a s d id n o t p r e v e n t the d e v e lo p m e n t o f

f i b r o p a p i l l o m a s on the sam e c a l f ( ta b le 23) n o r w a s

the r e g r e s s io n o f tho se f i b r o p a p i l l o m a s a c c o m p a n ie d

by the r e g r e s s io n o f p a p i l l o m a s ,

C) F ig u r e 14 show s the h i s t o l o g ic a l a p p e a ra n c e o f b o th

the le s io n s f r o m w h ic h B P V w as e x t r a c t e d a n d those


M a c r o s c o p ic a l l y , the le s io n s p r e s e n t as f i b r o p a p i l l -

o m a s on the bov ine te a t . T h e y v a r y w id e l y in s ize

f r o m < 1 c m to > 5 c m , o c c a s io n a l ly e x c e e d in g the

s iz e o f the te a t . T h e y m a y be p e d u n c u la te d b u t

m o r e c o m m o n ly a re s e s s i le . The le s io n s c o m m o n ly

have f r o n d s > 2 m m in le n g th b u t o c c a s io n a l l y the se

m a y ^ a b s e n t f r o m p a r t o r the w h o le s u r fa c e le a v in g a

s m o o th c o v e r in g o f r e l a t i v e l y n o r m a l e p i t h e l iu m .

In c o n t r a s t to the le s io n s r e p o r te d u n d e r E / f o l lo w in g ,

. 101 ,

iaisasb .

iiae

îM iF IG U R E 14 (a) H is t o lo g i c a l a p p e a ra n c e o f te a t

f i b r o p a p i l l o m a f r o m w h ic h B P V w a s

e x t r a c te d (b) T r a n s m i t t e d le s io n

sh o w in g f i b r o p a p i l l o m a on s k in o f c a l f

68893. T hese le s io n s a r e n o t

h i s t o l o g i c a l l y s e p a ra b le f r o m those

i l l u s t r a t e d in f i g u r e 15.

S ta in H & E . B a r = 1 m m .

. 102 .

T A B L E 23 - C H A L L E N G E O F IN O C U L A T E D C A L V E S B YC O N T A C T W IT H B P V I N F E C T E D C A L V E S .

C A L F L E S IO N T Y P E

a t i n o c u la t i o n (see t a b le § ^ s i te .

a t n o n - in o c u la t io n s i te s .

68882 A C

68886 E B

68888 B E

68891 D n o t a p p l ic a b le +

68893 C B & E

T F B S C ( i ) no le s io n s n o t a p p l ic a b le t

T F B S C ( i i ) no le s io n s no le s io n s

T F B M C no le s io n s no le s io n s

F BSC no le s io n s E

F B M C no le s io n s no le s io n s

L e s io n type d e s c r ib e d in M a t e r i a l s & M e th o d s as A - - E .

+ T hese c a lv e s w e re n o t c h a l le n g e d by c o n ta c t bu t w e r e k e p t i s o la te d .

. 103 .

even te a t f i b r o p a p i l l o m a w i t h o u t a p a p i l l o m a to u s

s u r fa c e , s t i l l show p a p i l lo m a to u s c h a n g e s a ro u n d

the c i r c u m f e r e n c e o f the le s io n . T h e r e a r e ,

h o w e v e r , o c c a s io n a l le s io n s w h ic h c a n n o t be

d is t in g u is h e d g r o s s l y o r h i s t o l o g i c a l l y f r o m

a n o g e n i ta l f i b r o p a p i l l o m a e x c e p t f o r t h e i r a n a to m ic a l

s i te . T e a t f i b r o p a p i l l o m a s a re g r o s s l y and

h i s t o l o g i c a l l y in d is t in g u is h a b le f r o m the n o r m a l

c u ta n e o u s f i b r o p a p i l l o m a s r e p o r t e d in D / b e lo w ,

a g a in , e x c e p t f o r a n a to m ic a l s i te o f o c c u r r e n c e .

M ic r o s c o p i c a l l y , f i b r o p a p i l l o m a s a r e c h a r a c t e r i s e d

by h y p e r k e r a t o s i s , a c a n th o s is and a c o n c u r r e n t

d e r m a l f i b r o m a , i n t im a t e l y in v o lv e d w i t h a

d is o r g a n is e d and s o m e t im e s a b s e n t s t r a t u m b a sa le

in e a r l y le s io n s . O n o c c a s io n s , t h i s le a d s to a re a s

o f u l c e r a t i o n - n o t ic e d as sp on tan e ou s b le e d in g on

c a l f 688 93 a t w e e k 12 p o s t i n o c u la t io n . The e a r l y

le s io n s a ls o show a r e a s o f a c a n th o s is w i t h a

t h i c k e n in g o f the s t r a t u m g r a n u lo s u m , th o u g h le s s

p ro n o u n c e d tha n in A o r B a b o v e . I n t r a n u c le a r

i n c lu s io n s ( b a s o p h i l i c ) a re r a r e l y seen in e a r l y

le s io n s w i t h t h e i r f i r s t a p p e a ra n c e 10 w e e k s p o s t

i n o c u la t io n b e c o m in g m o r e c o m m o n w i t h a d v a n c in g

t im e . A s le s io n s m a tu r e ,a c a n th o t ic e p i t h e l iu m is

t h r u s t u p w a rd s by c o n n e c t iv e t is s u e p a p i l l a e c o m p o s e d

e n t i r e l y o f f i b r o b la s t i c c e l ls w i t h n u m e r o u s v a s c u la r

c h a n n e ls . The f i r s t e v id e n c e o f a t t e m p te d r e je c t i o n

w a s n o t ic e d ( ta b le 22) in c a l f 68893 14 w e e k s p o s t

in o c u la t io n , b e c a m e m o r e p ro n o u n c e d a t 16 - 18

w e e k s w i t h the a c c u m u la t io n o f l a r g e n u m b e r s o f

l y m p h o c y te s b u t r e d u c e d a t w e e k 20 w i t h o u t

s u c c e s s f u l l y a f f e c t in g the p r o g r e s s iv e d e v e lo p m e n t

o f la r g e f i b r o p a p i l l o m a s . The p re s e n c e o f

d e v e lo p in g f i b r o p a p i l l o m a s d e r iv e d f r o m te a t le s io ns ,

and o b v io u s , though u n s u c c e s s fu l , e v id e n c e o f

, 1 0 4 ,

r e je c t io n , d id n o t p r e v e n t c a l f 68893 d e v e lo p in g

b o th p a p i l l o m a s and a n o g e n i ta l f i b r o p a p i l l o m a s as

a r e s u l t o f the c o n ta c t c h a l le n g e p a r t o f the

expe r i m e n t .

D) F ig u r e 15 show s the h is t o l o g i c a l a p p e a ra n c e o f bo th

the le s io n s f r o m w h ic h B P V w a s e x t r a c t e d and

those p ro d u c e d by t r a n s m is s io n to c a l f 68891

( ta b le 22). M a c r o s c o p ic a l l y , c u ta n e o u s

f i b r o p a p i l l o m a a r e in d is t in g u is h a b le f r o m those

p r e v io u s l y d e s c r ib e d in the l i t e r a t u r e (B a g d o n a s and

O ls o n , 1 9 5 3 ). U n l ik e te a t f i b r o p a p i l l o m a th e y a r e

r a r e l y s u b je c te d to s u f f i c i e n t r e g u la r t r a u m a to

cause p a r t i a l o r c o m p le te lo s s o f t h e i r p a p i l l o m a to u s

s u r f a c e . The le s io n s a re g e n e r a l l y in d is t in g u is h a b le

f r o m those d e s c r ib e d u n d e r C / above b u t can be

r e a d i l y s e p a ra te d g r o s s l y and h i s t o l o g i c a l l y f r o m

a n o g e n i ta l f i b r o p a p i l l o m a s by the p re s e n c e o f a

p ro n o u n c e d p a p i l l o m a to u s s u r fa c e w i t h f r o n d le n g th

> 2 m m . On t r a n s m is s io n , c a l f 68891 ( ta b le 22)

f i r s t show ed f i b r o p a p i l l o m a a t w e e k 8 p o s t

i n o c u la t io n w i t h the a c c o m p a n y in g p re s e n c e o f s m a l l

a r e a s o f ly m p h o c y te a c c u m u la t io n . B y w e e k 14, the

f i b r o p a p i l l o m a h ad been r e je c t e d f o l l o w in g the

i n f i l t r a t i o n o f la r g e m a s s e s o f ly m p h o c y te s w i t h

a s s o c ia te d n e c r o s is a nd c e l l dea th a t w e e k 10. C a l f

68891 w a s h e ld in i s o la t io n d u r in g the e x p e r im e n t ,

and i t d id n o t d e v e lo p le s io n s on a n y s k in s i te o th e r

tha n the i n o c u la t io n s i te s .

E ) F ig u r e 16 show s the h is t o l o g i c a l a p p e a ra n c e o f bo th

the le s io n s f r o m w h ic h B P V w a s e x t r a c t e d and those

p ro d u c e d by t r a n s m is s i o n to c a l f 68886 ( ta b le 22).

M a c r o s c o p i c a l l y , the le s io n s p r e s e n t as la r g e ,

s e s s i le o r m o r e c o m m o n ly p e d u n c u la te d c a u l i f l o w e r -

1 0 5 .

ïï-

m

F IG U R E 15 (a ) H is t o lo g i c a l a p p e a ra n c e o f c u ta n e o u s

f i b r o pa p i 11 orna f r o m w h ic h B P V w a s

e x t r a c te d (b) T r a n s m i t t e d le s io n

s h o w in g f ib r o p a p i l lo m a on s k in o f c a l f

68891. T h e se le s io n s a r e n o t

h i s t o l o g i c a l l y s e p a ra b le f r o m those

i l l u s t r a t e d in f i g u r e 14.

S ta in H & E . B a r = 1 m m .

106

F IG U R E 16 (a) H is t o lo g i c a l a p p e a ra n c e o f a n o - g e n i t a l

f i b r o p a p i l l o m a f r o m w h ic h B P V w a s

e x t r a c te d (b) t r a n s m i t t e d le s io n s h o w in g

f i b r o p a p i l l o m a on s k in o f c a l f 68886.

T h e se le s io n s a r e r e a d i l y s e p a ra b le

f r o m o th e r f i b r o p a p i l l o m a s by the

a b se n ce o f f r o n d s > 2 m m lo n g and by

t h e i r c a u l i f l o w e r - l i k e a p p e a r a n c e . N o te

the r e la t i v e a p p e a ra n c e o f the e p i t h e l iu m

w hen c o m p a r e d w i t h o th e r le s io n s

i l l u s t r a t e d h e r e . The m a jo r a p p e a ra n c e

is th a t o f a f i b r o m a w i t h s e c o n d a ry

e p i t h e l ia l c h a n g e s .

S ta in hi & E . B a r = 1 m m .

1

l i k e le s io n s . P a p i l l o m a t o s i s is re d u c e d o r a b s e n t

( f r o n d s C 2 m m ) g iv in g the a p p e a ra n c e in e a r l y

le s io n s o f a s m o o th s u r fa c e . O n in c i s io n , the

le s io n c o m p r is e s a w h i t i s h , f l e s h y f i b r o m a c o v e r e d

by a t h in e p i t h e l i a l l a y e r w h ic h in l a t e r le s io n s m a y

b e c o m e s l i g h t l y th ic k e n e d . H is t o l o g i c a l l y , the

le s io n s a re c h a r a c t e r i s e d by the d e v e lo p m e n t o f a

r a p id l y g r o w in g f i b r o m a i n t im a t e l y in v o lv e d w i t h the

i n d i s t i n c t s t r a t u m b a sa le o f an o th e r w is e n o r m a l

e p i t h e l i u m . O th e r d e r m a l s t r u c t u r e s a r e pushed

l a t e r a l l y a nd a p p e a r in c o n c e r t in a f o r m a d ja c e n t to

the m a in le s io n . E p i t h e l i a l r e te b r id g e s and h a i r

sh e a th s b e c o m e th in and g ive the a p p e a ra n c e o f

a r b o r i s a t i o n due to the d i s t o r t i v e e f f e c t s o f the

f i b r o m a . C e l ls in the f i b r o m a a r e p lu m p f i b r o b la s t s

w i t h open o v a l n u c le i o f te n c o n ta in in g s e v e r a l n u c le o l i .

P ro n o u n c e d c a p i l l a r y i n f i l t r a t i o n i s a f e a tu r e ,

a c c o m p a n ie d by the o c c a s io n a l a p p e a ra n c e o f r e d

b lo od c e l l s b e tw e e n t is s u e s p a c e s . I n the m a tu r e

le s io n , s l ig h t h y p e r k e r a t o s i s w i t h m o d e r a te

a c a n th o s is and i n t e r m i t t e n t p a r a k e r a t o s i s a r e seen .

O c c a s io n a l l y i n t r a n u c le a r in c lu s io n s a r e v i s i b l e , bu t

n o t as m a r k e d as in B / a bo ve . On t r a n s m is s io n

( ta b le 22) le s io n s w e r e f i r s t n o t ic e d 6 w e e k s p o s t

i n o c u la t io n a c c o m p a n ie d by s m a l l a c c u m u la t io n s o f

l y m p h o c y t e s . B y w e e k 10, la r g e a r e a s o f the

f i b r o m a c o n ta in e d m a s s iv e a c c u m u la t io n s o f

l y m p h o c y te s , w i t h a d ja c e n t e v id e n c e o f n e c r o s i s and

c e l l d e a th . B y w e e k 12, bo th the e p i t h e l i a l and

f i b r o m a to u s c o m p o n e n ts w e r e r e je c t e d , r e p la c e d by

a p p a r e n t ly n o r m a l e p i t h e l iu m and s c a r t i s s u e . The

d e v e lo p m e n t and r e g r e s s io n o f f i b r o p a p i l l o m a s on

c a l f 68886 d id n o t p r e v e n t the d e v e lo p m e n t and

p e r s is t e n c e o f p a p i l l o m a s as d e s c r ib e d u n d e r B /a b o v e

on the sa m e a n im a l .

. 1 0 8 .

F ) C a lv e s in o c u la te d w i t h c o n t r o l F B M C a n d F B S C

an d th o se in o c u la te d w i t h B P V t r a n s f o r m e d (T )

F B S C and T F B M C d e v e lo p e d no v i s i b l e le s io n s a t

any in o c u la t io n s i te in the s ix m o n th s p r i o r to the

c h a l le n g e s e c t io n o f the e x p e r im e n t . B io p s ie s a t

f o r t n i g h t l y i n t e r v a l s f a i l e d to show h i s t o l o g i c a l

e v id e n c e o f in o c u la te d c e l l g r o w th . The f i r s t b io p s y

a t 2 w e e k s in d ic a te d the p re s e n c e o f f i b r o b l a s t i c

c e l l s a t b o th F B S C and T F B S C c a rb o n la b e l le d

in o c u la t io n s i t e s .

3 . 3 . 2 C h a l le n g e

A . B y C o n ta c t

B e tw e e n tw o and e ig h t w e e ks p o s t in o c u la t io n , a l l

c a lv e s (e x c e p t 68891 and T F B S C ( i ) l i s t e d ta b le s

23 and 24) w e re h e ld in one pen . W e e k ly

e x a m in a t io n s re v e a le d the t r a n s m is s io n o f le s io n s

c la s s e d as B, C and E to n o n - in o c u la t io n s i te s

( m a in ly the h e a d ) . T a b le 23 l i s t s the le s io n typ e s

seen d u r in g th is s e c t io n o f the e x p e r im e n t . The two

t r a n s f o r m e d c e l l in o c u la te d c a lv e s (T F B S C ( i i ) and

T F B M C ) d id n o t d e v e lo p le s io n s , w h i le one (F B S C )

o f the tw o c o n t r o l c e l l in o c u la te d c a lv e s d e v e lo p e d

le s io n s in d is t in g u is h a b le f r o m E d e s c r ib e d a b o ve .

L e s io n s B & E w e r e each t r a n s m i t t e d to tw o c a lv e s

w h i le le s io n s C w a s t r a n s m i t t e d to one c a l f . L e s io n

A w a s n o t t r a n s m i t t e d by c o n ta c t to a n y c a l f and i t

s h o u ld be n o te d th a t a p p r o x im a t e ly h a l f o f the f i r s t

in o c u la t io n s c a r i f i c a t i o n l in e s u s in g B P V f r o m le s io n

A on c a l f 6888 2 f a i l e d to d e v e lo p le s io n s . T o ta l

l e s io n s iz e has n e v e r been seen to e x c e e d 1 c m in

d ia m e t e r e i t h e r u n d e r n a t u r a l c o n d i t io n s on the

b o v in e te a t o r e x p e r im e n t a l l y . T h e r e f o r e a f o u r

p lu s r a t in g as on ta b le s 22 and 26-was n o t a r e a s o n a b le

109 .

e x p e c ta t io n f o r le s io n A .

B . B y S c a r i f i c a t i o n

T a b le 24 l i s t s the r e s u l t s o f c h a l le n g e o f e a ch c a l f

by s c a r i f i c a t i o n u s in g B P V f r o m f i v e le s io n ty p e s

(A ~ E ) . The r e s u l t s can b e s t be s u m m a r is e d

a c c o r d in g to i n d iv id u a l c h a l le n g e s i t e s . W he n

c h a l le n g e d w i t h B P V f r o m le s io n A , o n ly one o f the

v i r u s i n o c u la t e d c a lv e s d e v e lo p e d le s io n s - c a l f

6 8 8 9 1 w h ic h had r e c e iv e d v i r u s f r o m le s io n D and

ha d been k e p t in i s o la t i o n . One o f the c e l l

in o c u la te d c a lv e s f a i l e d to d e v e lo p le s io n s , the

is o la t e d c a l f T F B S C ( i ) . C ha l le n g e w i t h B P V f r o m

le s io n B sh ow ed a s i m i l a r re s p o n s e w i t h o n ly v i r u s

in o c u la te d c a l f 68891 and c e l l in o c u la te d c a l f

T F B S C ( i ) s h o w in g re s p o n s e s d i f f e r e n t to the o th e r s .

C h a l le n g e w i t h B P V f r o m le s io n C / a g a in sh ow ed the

s u s c e p t ib i l i t y o f c a l f 68891 th o u g h w i t h a re d u c e d

re s p o n s e w i t h d e la y e d d e v e lo p m e n t o f s m a l l le s io n s

1 c m by w e e k 12 p o s t c h a l le n g e . W h i le le s io n

C / B P V c h a l le n g e d , c e l l in o c u la te d c a lv e s show ed

v a r ia b le r e s p o n s e s . T F B S C ( i ) and T F B S C ( i i )

f a i l e d to d e v e lo p le s io n s w h i le T F B M C d e v e lo p e d

s m a l l e a r l y le s io n s w h ic h r a p id l y r e g r e s s e d .

F B S C and F B M C c o n t r o l c a lv e s b o th d e v e lo p e d

le s io n s , th o u gh F B S C show ed a r e d u c e d re s p o n s e in

d e la y e d d e v e lo p m e n t and s m a l l s iz e le s io n s a t w e e k

12 . C h a l le n g e d w i t h B P V f r o m le s io n D , t h r e e o f

the f iv e v i r u s in o c u la te d c a lv e s re s p o n d e d w i t h the

e a r ly d e v e lo p m e n t and r e je c t i o n o f s m a l l ( <. 1 c m )

le s io n s w h i le tw o (6888 6 and 68893) sh ow ed no

re s p o n s e . T w o o f the th re e t r a n s f o r m e d c e l l

in o c u la te d c a lv e s d e v e lo p e d a re d u c e d re s p o n s e w i t h

e a r l y d e v e lo p m e n t o f s m a l l e r le s io n s a nd e a r l y

r e je c t i o n . One t r a n s f o r m e d c e l l in o c u la te d c a l f

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. 1 1 1 ,

(T F B S C ( i i ) ) f a i l e d to re s p o n d . One c o n t r o l c a l f

( F B M C ) sh ow ed c o m p le te s u s c e p t i b i l i t y w h i le the

o th e r , F B S C , w h ic h h ad re s p o n d e d to E in the c o n ta c t

c h a l le n g e p e r io d , f a i l e d to r e s p o n d to c h a l le n g e by

s c a r i f i c a t i o n . W h e n c h a l le n g e d w i t h B P V f r o m

le s io n E , tv/o c a lv e s re s p o n d e d . C a l f 68891 sh ow e d

e a r l y re s p o n s e w i t h le s io n s o f r e d u c e d s iz e w h i le the

c o n t r o l c e l l in o c u la te d c a l f F B M C w a s c o m p le t e ly

s u s c e p t ib le . C a l f F B S C had d e v e lo p e d le s io n E

f i b r o p a p i l l o m a s d u r in g the c o n ta c t c h a l le n g e p e r io d ,

3 . 3 . 3 E le c t r o n M ic r o s c o p y

T h in s e c t io n e x a m in a t io n f a i l e d to d e te c t the p re s e n c e

o f B P V in B P V in fe c te d F B M C and F B S C a t a n y t im e b eyo n d

the f i r s t p a s s a g e . E . M . e x a m in a t io n o f n e g a t i v e l y s ta in e d

p r e p a r a t io n s o f s p e n t c u l t u r e m e d iu m a ls o f a i l e d to d e te c t

B P V . A l l v i r u s p r e p a r a t io n s u s e d f o r a n im a l in o c u la t io n , and

s c ra p e s a m p le s o f t r a n s m i t t e d le s io n s on c a lv e s show ed the

p re s e n c e o f B P V in n e g a t i v e l y s ta in e d p r e p a r a t io n s w h e n

e x a m in e d u n d e r the E , M ,

3 . 3 . 4 P o s t M o r t e m E x a m in a t io n

D e ta i le d p o s t m o r t e m e x a m in a t io n d e te c te d the

p re s e n c e o f u p p e r a l i m e n t a r y p a p i l l o m a s in t h r e e o f the e ig h t

c a lv e s th a t w e r e n o t k e p t u n d e r i s o la t io n .

C a l f 68888 show ed p a p i l l o m a s on the s o f t p a la te , tongue

a nd o e s o p h a g u s ( f i g u r e 17 (b) )

C a l f F B M C sh ow ed p a p i l l o m a s on the b u c c a l m u c o s a and

the m u c o s a l i n in g the i n t e r n a l n a r e s

( f i g u r e 17 (a) ) and the m u c o c u ta n e o u s ju n c t io n .

C a l f T F B S C ( i i ) show ed p a p i l l o m a s on the o e s o p h a g u s .

C a l f 68888 a d d i t i o n a l l y show ed an e r o s iv e , g r a n u lo

m a to u s le s io n on the r u m in a i w a l l and c y s t i c h y p e r p la s ia o f

the g a l l b la d d e r w a l l .

. 1 1 2 .

1

â

F IG U R E 17 (a) F ib r o p a p i l l o m a p r e s e n t a t m u c o c u ta n e o u s

ju n c t i o n o f o r a l c a v i t y o f c a l f F B M C , th is

c a l f had f i b r o p a p i l l o m a s and p a p i l l o m a s

p r e s e n t bo th w i t h in the o r a l c a v i t y a nd on

the e x te r n a l cu ta n e o u s s u r fa c e o f the fa c e ,

(b) P a p i l l o m a on w a l l o f o e s o p h a g u s o f c a l f

68888, th is c a l f a ls o had p a p i l l o m a s on the

p a la te , tongue and o r o p h a r y n x .

S ta in I I & E . B a r = 1 m m .

113

H a e m a to lo g i c a l e x a m in a t io n o f s e q u e n t ia l b lo o d s a m p le s w a s

u n r e m a r k a b le . A p p e n d ix C r e c o r d s the h a e m a t o lo g ic a l

r e s u l t s in d e ta i l .

3 . 4 D is c u s s io n

T h e i s o la t i o n o f B P V f r o m and the d e te c t io n o f th re e

m o r p h o lo g i c a l l y s e p a ra b le te a t le s io n s have been r e p o r t e d

p r e v io u s l y (C h a p te r 3 ). H o w e v e r the t r a n s m is s io n o f th e se le s io n s

to o th e r p a r t s o f the in te g u m e n t e x p e r im e n t a l l y r e p r e s e n t s a new

f in d in g . O n t r a n s m is s io n , B P V e x t r a c te d f r o m one le s io n type

p ro d u c e d le s io n s o f the sa m e type on a n o th e r a n a t o m ic a l s i t e . The

e x is te n c e o f p a p i l l o m a s a nd the e x t r a c t i o n o f B P V f r o m th e m has

been r e p o r t e d b e fo re ( B a r t h o ld e t a l . , 1974), h o w e v e r a t t e m p ts a t

e x p e r im e n t a l t r a n s m is s io n and, im m u n o lo g i c a l c h a r a c t e r i s a t i o n o f

the v i r u s u s in g m e th o d s s ta n d a r d f o r B P V w e r e u n s u c c e s s fu l .

B a r t h o ld e t a l . , (1974 ) n o te d the te n d e n c y o f t u m o u r s to p e r s i s t ,

r a t h e r tha n r e g r e s s w h ic h o c c u r r e d w i t h c a l f 68888 ( ta b le 22) in the

e x p e r im e n t s r e p o r t e d h e r e . S i m i l a r l y , p a p i l l o m a s w e r e r e p o r t e d

to o c c u r c o n c u r r e n t l y o r s u b s e q u e n t ly on a n im a ls w i t h t y p i c a l f i b r o

p a p i l l o m a s . T h is c h a p te r a ls o r e c o r d s the d e v e lo p m e n t and

r e g r e s s io n o f f i b r o p a p i l l o m a s on c a lv e s 68888 and 68882 s h o w in g

c o n c u r r e n t p a p i l l o m a s and r i c e g r a in le s io n s r e s p e c t i v e l y w h ic h d id

n o t r e g r e s s .

R ic e g r a in le s io n s a r e m a c r o s c o p ic a l l y and h i s t o l o g i c a l l y

s i m i l a r to f o c a l e p i t h e l ia l h y p e r p la s ia ( P r a e t o r i u s - C la u s e n , 1972)

w h ic h o c c u r s in the h u m a n o r a l c a v i t y , and in w h ic h a p ap o va v i r u s

has been d e te c te d ( P r a e t o r i u s -C la u s e n and W i l l i s , 1 971 ), The

le s io n s t r a n s m i t t e d to c a l f 68882 p e r s is t e d f r o m w e e k 6 to w e e k 20

p o s t in o c u la t io n and r e j e c t i o n w a s n o t a c c o m p a n ie d by the m a s s iv e

ly m p h o c y te i n f i l t r a t i o n a s s o c ia te d w i t h r e g r e s s io n o f f i b r o p a p i l l o m a s

O f the t h r e e f i b r o p a p i l l o m a t r a n s m i t t e d c a lv e s , o n ly one in o c u la te d

w i t h B P V f r o m a n o g e n i ta l f i b r o p a p i l l o m a s p ro d u c e d le s io n s

m a c r os c o p i c a l l y and h i s t o l o g i c a l l y s e p a ra b le f r o m the o th e r tw o ,,

c o n f i r m in g e a r l i e r o b s e r v a t io n s on f i e ld case l e s io n s . H o w e v e r ,

114

bo th th e a n o g e n i ta l and the c u ta n e o u s f i b r o p a p i l l o m a s sh o w e d an

e a r l y a nd s u c c e s s f u l r e je c t i o n re s p o n s e a t w e e k s 8 - 1 0 p o s t

i n o c u la t io n w h i le c a l f 68893 show ed a d e la y e d r e j e c t i o n re s p o n s e

1 6 - 1 8 w e e k s p o s t in o c u la t io n w h ic h w as u n s u c c e s s fu l r e s u l t i n g in

the p e r s is t a n c e o f l e s io n s t i l l p o s t m o r t e m a t 24 w e e k s . The

abse n ce o f E M de te c ta .b le B P V in t r a n s f o r m e d o r c o n t r o l F B S C

and F B M C w a s c o n f i r m e d in v iv o by the a bse n ce o f l e s io n

d e v e lo p m e n t d u r in g the s ix m o n th s p o s t i n o c u la t io n .

C o m b in in g the r e s u l t s o f c h a l le n g e by c o n ta c t and s c a r i f i c a t i o n ,

the f o l l o w in g p o in ts m a y be m a d e .

1) A n i m a l s s h o w in g p a p i l l o m a s o r r i c e g r a in l e s io n s r e m a in

s u s c e p t ib le to t r a n s m is s io n o f f i b r o p a p i l l o m a s . R e je c t io n

o f f i b r o p a p i l l o m a s is n o t a c c o m p a n ie d by r e j e c t i o n o f

p a p i l l o m a s o r r i c e g r a in le s io n s .

2) A n i m a l s s h o w in g e i t h e r p a p i l l o m a s o r r i c e g r a in le s io n s a re

im m u n e to c h a l le n g e by s c a r i f i c a t i o n o f B P V f r o m bo th le s io n

ty p e s ( c a lv e s 68882, 68886, 68888 and 688 93) . H o w e v e r

a n im a ls n o t s h o w in g e i t h e r le s io n type r e m a in s u s c e p t ib le to

c h a l le n g e by B P V f r o m b o th le s io n typ e s ( c a lv e s 68891,

T F B S C ( i i ) , T F B M C , F B S C , F B M C ) . O n ly one c a l f ,

in o c u la te d w i t h T F B S C ( i ) show ed r e s is t a n c e to c h a l le n g e by

b o th B P V is o la t e s w i t h o u t s h o w in g p r i o r le s io n s .

3) W i t h in the f i b r o p a p i l l o m a g ro u p ( le s io n s C, D, E ) , p r i o r

i n f e c t i o n by B P V f ro m , a n o g e n i ta l f i b r o p a p i l l o m a s (E ) w a s

f o l lo w e d by r e s is t a n c e to c h a l le n g e by s c a r i f i c a t i o n o f B P V

f r o m le s io n C in c a lv e s 68882, 68886, 68888 and 68893 ( w h ic h

had e a r l i e r d e v e lo p e d le s io n C and E f o l l o w in g in o c u la t io n

and c o n ta c t c h a l le n g e ) C a l f F B S C w h ic h d e v e lo p e d le s io n E

in the c o n ta c t c h a l le n g e s tage show ed a re d u c e d re s p o n s e to

le s io n C s c a r i f i c a t i o n c h a l le n g e . C a l f F B M C w a s c o m p le t e ly

s u s c e p t ib le to c h a l le n g e by s c a r i f i c a t i o n o f a l l f i b r o p a p i l l o m a

d e r i v e d B P V (C, D, E ) . O f the th r e e c a lv e s w h ic h r e c e iv e d

le s io n D t r a n s f o r m e d c e l l s , T F B S C ( i ) a n d T F B S C ( i i ) w e r e

. 115

r e s i s t a n t to c h a l le n g e by bo th le s io n C and E w h i le c a l f

T F B M C show ed r e s is ta n c e to le s io n E and a re d u c e d re s p o n s e

to le s io n C . F i n a l l y is o la te d c a l f 68891 w h ic h d e v e lo p e d

le s io n D w a s s u s c e p t ib le bu t w i t h a re d u c e d re s p o n s e to B P V

f r o m a l l th r e e f i b r o p a p i l l o m a le s io n s . H o w e v e r re s p o n s e

to le s io n D w a s m o r e r a p id l y r e je c te d . C h a l le n g e by le s io n

D B P V s c a r i f i c a t i o n r e s u l t e d in no re s p o n s e in c a lv e s 68886,

68893, T F B S C ( i ) and F B S C and a re d u c e d re s p o n s e in a l l

o th e r c a lv e s e x c e p t F B M C w h ic h r e m a in e d f u l l y s u s c e p t ib le .

4) C o n t r o l c e l l in o c u la te d c a lv e s , F B S C and F B M C , w e re

s u s c e p t ib le to c h a l le n g e s ta g e s o f the e x p e r im e n t .

T r a n s f o r m e d c e l l in o c u la te d c a lv e s w e re e i t h e r r e s i s t a n t o r

d e v e lo p e d a r e d u c e d re s p o n s e to c h a l le n g e by B P V f r o m the

th r e e f i b r o p a p i l l o m a le s io n ty p e s .

T h e se e x p e r im e n t s s u g g e s t th a t th e re a r e tw o b ro a d B P V

g ro u p s , B P V f r o m p a p i l l o m a and r i c e g r a in le s io n s and B P V f r o m

f i b r o p a p i l l o m a s . W i t h in the se two g ro u p s th e r ^ a r e g r o s s ,

m i c r o s c o p i c , d e v e lo p m e n ta l d i f f e r e n c e s w h ic h a r e r e p r o d u c ib le by

e x p e r im e n t a l t r a n s m is s io n . A n i m a l r e s is ta n c e to one b ro a d g ro u p

does n o t in f lu e n c e r e s is t a n c e to the o th e r g ro u p . The use o f c e l l s

t r a n s f o r m e d in v i t r o by f i b r o p a p i l l o m a d e r iv e d B P V in c r e a s e s

r e s is t a n c e t o c h a l le n g e by f i b r o p a p i l l o m a d e r iv e d v i r u s e s b u t n o t

B P V e x t r a c t e d f r o m p a p i l l o m a o r r i c e g r a in le s io n s .

T h e s e f in d in g s m a y p a r t l y e x p la in the w id e l y v a r y in g r e s u l t s

o f B P V v a c c in a t io n r e p o r t e d in the l i t e r a t u r e , ( r e v ie w by O ls o n

e t a l . l 9 69). The f in d in g o f a l im e n t a r y p a p i l l o m a s in t h r e e o f

e ig h t in c o n ta c t c a lv e s a t p o s t m o r t e m w a s u n e x p e c te d a l th o u g h the

s u g g e s t io n has been m a d e e a r l i e r (C h a p te r 2; J a r r e t t e t a l , , 1978)

th a t te a t p a p i l l o m a t o s i s m a y le a d to a l im e n t a r y p a p i l l o m a s . A

s u rv e y o f o v e r 7, 000 c a t t le show ed an o v e r a l l p r e v a le n c e o f

a l im e n t a r y p a p i l l o m a s o f 19% ( J a r r e t t e t a l . , 1978). The n u m b e r o f

a n im a ls u s e d in the e x p e r im e n ts r e p o r t e d in t h i s p a p e r a re s m a l l .

and the r e s u l t s m u s t n e c e s s a r i l y be t r e a te d as p r e l i m i n a r y ,

, 1 1 6 .

%

F IG U R E 18 P h o to g ra p h s h o w in g m a c r o s c o p ic

d i f f e r e n c e be tw een t r a n s m i t t e d a n o g e n i ta l

f i b r o p a p i l l o m a (a) and te a t f i b r o p a p i l l o m a

(b) c a l f F B M C . N o te s m o o th s u r fa c e o f

(a) as c o n t r a s te d w i t h p a p i l l o m a to u s

s u r fa c e o f b) B a r = 2 c m .

. 117 ,

F u r t h e r in v iv o and in v i t r o w o r k u s in g B P V f r o m s in g le c a s e ,

s in g le typ e le s io n s m u s t be done to f u l l y e lu c id a te the d i f f e r e n c e s

b e tw e e n v a r io u s B P V e x t r a c t s .

S U M M A R Y

B o v in e p a p i l l o m a v i r u s (B P V ) w a s e x t r a c te d f r o m f i v e c a t t le

each a f fe c te d w i t h o n ly one o f f i v e m o r p h o lo g i c a l l y s e p a ra b le le s io n

ty p e s . W hen in o c u la te d in to e x p e r im e n t a l c a lv e s e i t h e r by

s c a r i f i c a t i o n o r i n t r a d e r m a l in je c t io n , the B P V e x t r a c t s p ro d u c e d

le s io n s m a c r o s c o p ic a l l y and m ic r o s c o p i c a l l y s i m i l a r to those f r o m

w h ic h i n d i v id u a l e x t r a c t s w e r e m a d e . F o e ta l b o v in e c e l l s ,

t r a n s f o r m e d in v i t r o w i t h B P V , f a i l e d to p ro d u c e f i b r o m a s ,

f i b r o p a p i l l o m a s o r p a p i l l o m a s w h e n in o c u la te d in to e x p e r im e n t a l

c a lv e s . W h e n v i r u s a nd B P V t r a n s f o r m e d c e l l in o c u la te d c a lv e s ,

w e r e c h a l le n g e d w i t h the f iv e o r i g i n a l B P V e x t r a c t s , a d i f f e r e n t i a l

im m u n i t y w a s d e m o n s t r a te d , w h i le c o n t r o l c a lv e s w e r e

s u s c e p t ib le to a l l e x t r a c t s . P o s t m o r t e m e x a m in a t io n r e v e a le d

the p re s e n c e o f u p p e r a l im e n t a r y t r a c t p a p i l l o m a s in th re e o f

e ig h t c a lv e s f o r m in g one g ro u p . T hese r e s u l t s s u g g e s ts tha t

d i f f e r e n t B P V 's e x is t , c a u s in g m o r p h o lo g i c a l l y s e p a ra b le le s io n

ty p e s . T h e r e m a y be a d d i t io n a l B P V v a r ia n t s c a u s in g

f i b r o p a p i l l o m a s o f the te a t and a n o g e n i ta l r e g io n s o f c a t t le . The

i n o c u la t io n o f B P V t r a n s f o r m e d fo e ta l b o v in e c e l l s c o n f e r r e d a

r e la t i v e im m u n i t y to l a t e r c h a l le n g e w i t h som e b u t n o t a l l B P V

e x t r a c t s .

116

C H A P T E R 4

IN V IV O E F F E C T S O F B O V IN E P A P I L L O M A V IR U S ;

T H E T U M O R IG E N IC IT Y IN A T H Y M IC N U D E M I C E O F B O V IN E

P A P I L L O M A V IR U S A N D B P V T R A N S F O R M E D F O E T A L

B O V IN E S K IN C E L L S .

4. 1 I n t r o d u c t i o n

B o v in e P a p i l l o m a V i r u s (B P V ) has been sh ow n in v i t r o to

t r a n s f o r m b o v in e c e l l s ( B la c k e t a l . , 1963; T h o m a s e t a l . , 1963) and

p r i m a r y e m b r y o n ic c e l l c u l t u r e s f r o m C 3 H /e B , C 5 7 / B L and

B A L B / c m ic e (T h o m a s e t a l . , 1964 ). In v i t r o t r a n s f o r m a t i o n by

B P V has been r e p o r t e d u s in g fo e ta l bov ine c u l t u r e s d e r iv e d f r o m

s k in , p a la te , m e n in g e s and c o n ju n c t iv a (C h a p te r 7 ) . In v i v o , B P V

i s t u m o r ig e n ic in a w id e r ra n g e o f s p e c ie s than o th e r p a p i l l o m a

v i r u s e s . I n t r a c r a n i a l i n je c t i o n o f B P V p ro d u c e s m e n in g io m a s in

c a lv e s and f i b r o b l a s t i c i n t r a c r a n i a l n e o p la s m s in the h a m s te r

( G o rd o n a nd O ls o n , 1968; R o b l e t a l . , 1972). S u b cu ta n e o u s

i n je c t i o n o f B P V p r o d u c e s f i b r o m a s in C 3H / e B m ic e a nd h a m s te r s

( F r i e d m a n e t a 1. , 1963; C h e v i l le , 1966; R o b l and O ls o n , 1968), and

i n t r a d e r m a l i n je c t i o n o f B P V in the h o rs e in d u c e s a c o n n e c t iv e

t is s u e t u m o u r s i m i l a r to e q u in e s a r c o id , n a t u r a l l y o c c u r r i n g ca se s

o f w h ic h have been sh ow n to c o n ta in B P V v i r u s - s p e c i f i c D N A

se q u e n ce s (O ls o n and C o o k , l 9 5 l ; L a n c a s te r e t a l . , 1977). S e v e ra l

r e p o r t s s u g g e s t th a t B P V is t r a n s m is s ib l e to m a n , (S c h u l tz , 1908;

F r e n z , 1941; G r e n ie r e t a l . , 1976) a nd th is is s u p p o r te d by the

o b s e r v a t io n th a t p e r s o n s in c e r t a i n o c c u p a t io n s s u ch as m e a t

h a n d le r s a re a t h ig h r i s k o f d e v e lo p in g w a r t s ( P e r e l and L u m p k in ,

1 9 7 6), T h is c h a p te r d e s c r ib e s the r e s u l t s o f i n o c u la t io n o f B P V ,

B P V t r a n s f o r m e d c e l l s and u n t r a n s f o r m e d c e l l s in to nude m ic e .

4. 2 M a t e r i a l s a nd M e th o d s

4 . 2 . 1 C e l l 6

The s k in o f a 60 c m . n e a r t e r m b o v in e fo e tu s w a s

l i g h t l y s c ra p e d w i t h a s t e r i l e s c a lp e l b la d e . C o l le c t e d

. 1 1 9 .

s c ra p in g s w e r e p la c e d in an 8oz . m e d ic a l f l a s k in 20 m l

b a s a l E ag les m e d iu m ( B M E ) w i t h 20% added f o e t a l b o v in e

s e r u m (F B S ) . The f i r s t s u b d iv is io n o f c e l l s o c c u r r e d 10

d ays l a t e r , and s u b s e q u e n t ly a t w e e k ly i n t e r v a l s u s in g B M E

w i t h 15% F B S . C u l t u r e s in the f o u r t h p assa g e w e re in fe c te d3. 5

in s u s p e n s io n w i t h 1 0 - ’ d i l u t i o n o f B P V (h ig h e s t d i lu t io n

s h o w in g 100% t r a n s f o r m a t i o n - see l a t e r ) as d e s c r ib e d

e ls e w h e re (C h a p te r 7) and the r e s u l t a n t t r a n s f o r m e d

c u l t u r e s f r o m p a ssa g e 12 w e r e u sed to in o c u la te nude m ic e .

P a r a l l e l u n t r a n s f o r m e d c u l t u r e s a t pa ssa g e 12, d e r iv e d f r o m

the sam e fo e tu s s e r v e d as c o n t r o ls f o r nude m o u s e

in o c u la t io n . P a i r e d B P V t r a n s f o r m e d and c o n t r o l f o e ta l

b o v in e s k in c e l l (F B S C ) c u l t u r e s w e re m a in ta in e d in v i t r o

t h r o u g h o u t the e x p e r im e n t . A t r e g u la r i n t e r v a l s , a s a m p le

o f b o th c e l l c u l t u r e s w as g ro w n on a g la s s s l id e p la c e d in a

10 c m . d ia m e t e r p la s t i c p e t r i d is h f o r e x a m in a t io n to c h e c k

m a in te n a n c e o f t r a n s f o r m e d p h e n o ty p e .

4 , 2 . 2 E x a m in a t io n o f C e l ls

C e l ls g ro w n on g la s s s l id e s w e r e s ta in e d w i t h

^ ie m s a a f t e r m e th a n o l f i x a t i o n as d e s c r ib e d e ls e w h e re

(C h a p te r 7). C e l ls w e r e e x a m in e d m a c ro s c o p ic a l l y , and

u n d e r a L e i t z l i g h t m ic r o s c o p e .

4 . 2 . 3 V i r u s

B P V w a s e x t r a c te d u s in g f i b r o p a p i l l o m a s c o l le c te d

f r o m a s in g le f i e l d case in fe c te d w i t h c u ta n e o u s f i b r o p a p i l l o

m a s (C h a p te r 3). T e c h n iq u e s used f o r e x t r a c t i o n have been

d e s c r ib e d e ls e w h e re (C h a p te r 7).

4 . 2 . 4 T r a n s f o r m a t i o n A s s a y

Pali r e d s a m p le s o f t r a n s f o r m e d and c o n t r o l F B S C

c u l t u r e s w e re a s s a y e d u s in g B P V d e s c r ib e d above to

d e te r m in e the L o g ^ ^ T r a n s f o r m in g D ose 50% (L o g T D ^ ^ )

( C h a p te r 7 ).

, 120 ,

4 . 2 . 5 N ude M ic e

S ix ty f i v e - B A L B / c r a n d o m o u tb r e d c r o s s 3 - 4 w e e k

o ld nude m ic e w e r e p u r c h a s e d f r o m O la c P ty L t d . T h e m ic e

w e r e r a n d o m ly d iv id e d in to th re e g ro u p s o f 20 a n d one g ro u p

o f f i v e a n im a l s . W i th in 24 h r s. a f t e r a r r i v a l a n im a ls w e r e

in o c u la te d w i t h P B SC, B P V t r a n s f o r m e d F B S C and B P V .

One 0. 1 m l in o c u la t io n w a s m a d e s u b c u ta n e o u s ly o v e r the r i b

cage on each s id e o f each a n im a l . A l l i n o c u la t io n s c o n ta in e d

l O m g / m l o f f i l t e r e d , s t e r i l i s e d a c t i v a te d c h a r c o a l w h ic h w a s

added, to m a r k the s i te o f i n je c t io n . C e l l i n o c u la t io n s w e r e

c a r r i e d o u t u s in g t h r i c e P B S r in s e d s u s p e n s io n s in PBS a t a7

c o n c e n t r a t io n o f 1 0 c e l l s / m l . T h us a l l a n im a ls r e c e iv e d

lO ^ c e l l s p e r i n o c u la t io n . T he v i r u s s u s p e n s io n w a s a 10%

w / v h o m o g e n a te f r o m s in g le case p a p i l l o m a s c l a r i f i e d by

c e n t r i f u g a t i o n f o r 30 m in a t 1 0 ,0 0 0 r . p . m . in an SW27 r o t o r ,

w i t h added 10 m g / m l a c t i v a te d c h a r c o a l . F iv e nude m ic e

w e r e n o t in o c u la te d and s e rv e d as u n t r e a te d c o n t r o ls .

4 . 2 . 6 H i s to p a th o lo g y

B e c a u s e o f the r e l a t i v e l y s h o r t l i f e span o f nude

m ic e h e ld u n d e r n o r m a l l a b o r a t o r y c o n d i t io n s , a n im a ls

w e r e e i t h e r s a c r i f i c e d in e x t r e m is o r d ie d n a t u r a l l y . E a c h

nude m o u s e w a s s u b je c te d to a d e ta i le d p o s t - m o r t e m

e x a m in a t io n . I n t e r n a l o rg a n s w e re r e m o v e d th r o u g h a

v e n t r a l m id l i n e i n c i s io n . T he w h o le c a r c a s e and o rg a n s

w e r e f i x e d f o r one w e e k in 10% f o r m o l s a l in e , b e fo re f i x e d

s a m p le s w e r e r e m o v e d f o r f u r t h e r p r o c e s s in g . I r r e s p e c t i v e

o f t r e a t m e n t , each a n im a l p r o v id e d a s e c t io n o f i n o c u la t io n

s i te , lu n g , l i v e r , s p le e n , k id n e y and a n y o th e r t is s u e s h o w in g

g r o s s p a th o lo g ic a l c h a n g e s . S e c t io n s w e r e r o u t i n e l y s ta in e d

w i t h H & E a nd w i t h a p p r o p r ia t e s p e c ia l s ta in s w h e n the need

a ro s e .

121 .

4 . 2 . 7 E le c t r o n M ic r o s c o p y

T u m o u r s f r o m te n nude m ic e w e r e e x c is e d a n d

i m m e d ia t e l y p la c e d i n 10% w / v P B S . T u m o u r t i s s u e w a s

h o m o g e n is e d u s in g a S i l v e r son b le n d e r a n d s u b je c te d to

d i f f e r e n t i a l c e n t r i f u g a t io n o u t l i n e d f o r v i r u s p r e p a r a t io n

e ls e w h e r e ( C h a p te r 7 ). S a m p le s o f c ru d e h o m o g e n a te

w e r e n e g a t i v e l y s ta in e d w i t h p h o s p h o tu n g s t ic a c id and

e x a m in e d f o r the p r e s e n c e o f B P V u n d e r the e le c t r o n

m ic r o s c o p e . S a m p le s f r o m CsC i^, g r a d ie n t p u r i f i e d

s u s p e n s io n s a ro u n d s p e c i f i c g r a v i t y o f 1 ,3 6 w e r e s i m i l a r l y

e x a m in e d .

A l l used m e d iu m f r o m t r a n s f o r m e d a nd c o n t r o l

F B S C c u l t u r e s w a s s e p a r a te ly c o l le c te d , p r e c ip i t a t e d a t

4 °C w i t h 50% ad d e d s a tu r a te d ( N H ^ j^ - S O ^ r e - s u s p e n d e d in

P BS and a g a in s u b je c te d to d i f f e r e n t i a l c e n t r i f u g a t i o n b e fo re

C sC 1 ^ g r a d ie n t p u r i f i c a t i o n and E M e x a m in a t io n f o r the

p re s e n c e o f B P V ,

T r a n s f o r m e d and c o n t r o l F B S C c u l t u r e s w e re

g ro w n on 5 c m p la s t i c p e t r i d is h e s f o r t h r e e d a y s , the m e d iu m

w a s r e m o v e d and c e l l s w e r e f i x e d in g lu ta r a ld e h y d e in s i t u .

A f t e r 24 h r s . c e l l s w e r e r e m o v e d u s in g a r u b b e r p o l i c e m a n

and f u r t h e r p r o c e s s e d f o r t h in s e c t io n e le c t r o n m ic r o s c o p y .

S a m p le s o f one tu m o u r p ro d u c e d by t r a n s f o r m e d F B S C

in o c u la t io n , w a s f i x e d in g lu ta r a ld e h y d e and f u r t h e r p r o c e s s e d

f o r t h in s e c t io n e le c t r o n m ic r o s c o p y .

4 . 2 . 8 A t t e m p t s a t In V iv o and In V i t r o P r o p o g a t io n o fT u m o u r s .

S ix t u m o u r s w e r e e x c is e d u n d e r s t e r i l e c o n d i t io n s

and r in s e d th r e e t im e s in B M E . T h re e o f the t u m o u r s w e re

c u t up in to s m a l l p ie c e s and p la c e d in an 8 o z . m e d ic a l f l a s k

w i t h 20 m l o f B M E w i t h 20% added F B S . T h e o th e r th r e e

tu m o u r s w e r e i n d i v i d u a l l y t r e a te d f o r t h r e e m in u te s in a .

S to m a c h e r b e fo re s i m i l a r l y b e in g p la c e d in to 8 o z . m e d ic a l

1 2 2 ,

f l a s k s w i t h 2Ü m l B M E w i t h 20% added F B S . B o t t le s w e re

e x a m in e d a t w e e k ly i n t e r v a l s f o r e v id e n c e o f c e l l g r o w th .

T w o tu m o u r s w e r e e x c is e d u n d e r s t e r i l e

c o n d i t io n s , t r e a te d in a S to m a c h e r f o r t h r e e m in u te s in 2 m l

B M E . The r e s u l t a n t c e l l s u s p e n s io n w as in o c u la te d

s u b c u ta n e o u s ly in to f i v e nude m ic e a f t e r a d d i t io n o f 1 0 m g / m l

o f a c t i v a te d c h a r c o a l . A n i m a l s w e r e o b s e r v e d f o r e v id e n c e

o f t u m o u r d e v e lo p m e n t .

4. 3 R e s u l t s

T a b le 25 s u m m a r is e s the e f fe c ts o f v a r io u s

t r e a t m e n t s on nude m ic e . No s ig n i f i c a n t d i f f e r e n c e s

o c c u r r e d b e tw e e n the m e a n s u r v i v a l t im e s o f t r a n s f o r m e d

F B S C , F B S C in je c t e d and c o n t r o l a n im a l s . H o w e v e r the

m e a n s u r v i v a l t im e o f the B P V in fe c te d nude m ic e w as

s ig n i f i c a n t l y l o w e r ( P < 0 . 0 1 ) . The d i s t r i b u t i o n o f m a jo r

le s io n s on an o rg a n by o r g a n b a s is w a s s i m i l a r in a l l t h r e e

g r o u p s .

A l l nude m ic e in o c u la te d w i t h B P V t r a n s f o r m e d

F B S C show ed c e l l p r o l i f e r a t i o n o u t o f the c a r b o n la b e l le d

in je c t i o n s i t e s . 45% o f these t u m o u r s r e s u l t e d in in v a s io n

o f a d ja c e n t t i s s u e s ( F ig u r e 20). Two a n im a ls i n the

u n t r a n s f o r m e d F B S C in o c u la te d g ro u p s h o w e d s o m e c e l l

o u tg r o w th , f r o m in je c t i o n s i te s , b u t th is w a s l i m i t e d and

n o n - in v a s iv e . One B P V in o c u la te d a n im a l sh ow ed an

i n f l a m m a t o r y re s p o n s e a t the in je c t io n s i te , h o w e v e r no

B P V in o c u la te d a n im a l show ed a n y e v id e n c e o f t u m o u r

p ro d u c t i o n .

F ig u r e 1 9 i l l u s t r a t e s the d i f f e r e n c e s b e tw e e n the

th r e e t r e a t m e n t g ro u p s g r a p h ic a l l y . T h e r e w a s a r a p id

g r o w th o f t u m o u r s f o r the f i r s t 30 days p o s t i n o c u la t io n .

T h e r e a f t e r , the t u m o u r s p r o g r e s s i v e l y c h an g ed to p ro d u c e

w e l l d i f f e r e n t i a t e d f i b r o m a s (see F ig u r e 20).

. 123 .

T A B L E 2 5 : R E S U L T S O F S /C I N O C U L A T IO N O F B P V ,B P V T R A N S F O R M E D F BSC A N D U N T R E A T E D F B S C IN T O N U D E - M I C E .

G R O U P A B C C o n t r o l

N u m b e r o f Nude, 6 , 6 . -1

5M ic e in o c u la te d w i t h

10 B P V t r a n s f o r m e d

F B S C .

10 F B S C 10 B P V n o th in g

M e a n s u r v i v a l37.8 - 4.4 33.6 t 3.0 2 1 .9 ^ 2. 5 40.2 i 8. 7t im e

+ S . E . (d a ys )

N u m b e ra n im a ls s h o w in g g r o w th a t i n je c t i o n s i te

N u m b e r o f a n im a lss h o w in g t u m o u r

20 2 i 0

in v a d in gs u r r o u n d in gt is s u e s

O rg a n in v o lv e d as m a jo r c a use o f d e a th .

9 0 0 0

L i v e r 45% 35% 40%L u n g 40% 45% 40%M ix e d 5% 20% 20%

124 .

X B P V t r a n s f o r m e d F B S C

© U n t r a n s f o r m e d F B S C

o B P V in o c u la te d

Q

Q

uoa0

rdPh

X

X X

XX

QOOÔ

Xo X

X X ^ X

J -------------- L

10 20 30 40 50 60 70

D a y s p o s t - i n o c u la t io n

80 90

FIGURE 19 : Nude m o u s e t u m o u r g ro w th r e la t e d to t im e p o s t

i n o c u la t io n .

125

F ig u r e 2 0 i l l u s t r a t e s these f e a tu r e s w i t h lo w p o w e r

p h o to m ic r o g r a p h s o f t u m o u r s f r o m tw o a n im a ls w h ic h d ie d

o r w e r e s a c r i f i c e d a t d i f f e r e n t t im e s p o s t - i n o c u l a t i o n .

C e i l type s in the o r i g i n a l in o c u lu m and in the

e a r l i e r , r a p id l y g r o w in g t u m o u r s w e r e s i m i l a r and l a r g e l y

c o m p r is e d f i b r o b la s t s as w e l l as a s m a l l e r p r o p o r t i o n o f

e p i th e l io d c e l l s w h ic h , w h e n u s in g s p e c ia l s ta in s , r e s e m b le d

k e r a t i n o c y te s . H ig h p o w e r p h o to m ic r o g r a p h s o f the se c e l l

ty p e s a p p e a r in F ig u r e 21.

D e ta i le d h is t o p a th o lo g ic a l e x a m in a t io n o f a l l

m a jo r i n t e r n a l o rg a n s f a i l e d to d e te c t any s ig n o f t u m o u r

m é ta s ta s é s o r p r i m a r y t u m o u r d e v e lo p m e n t . E l e c t r o n

m ic r o s c o p ic e x a m in a t io n o f h o m o g e n is e d t r a n s f o r m e d F B S C ,

F B S C and ten nude m o u s e t u m o u r s f a i l e d to d e te c t the

p re s e n c e o f B P V p a r t i c l e s in n e g a t i v e ly s ta in e d p r e p a r a t io n s .

E M e x a m in a t io n o f C s C l ^ p u r i f i e d sp e n t m e d iu m c o l le c te d

f r o m p a r a l l e l c e l l c u l t u r e s , n e g a t iv e ly s ta in e d , a ls o f a i l e d

to d e te c t B P V . T h in s e c t io n e le c t r o n m ic r o s c o p y f a i l e d t o ’

d e te c t B P V p a r t i c l e s in t r a n s f o r m e d F B S C , F B S C and nude

m o u s e t u m o u r s .

A t t e m p t s to p ro p a g a te nude m o u s e tu m o u r s

in v i t r o and in v iv o w e r e n o t s u c c e s s fu l i r r e s p e c t i v e o f the

t r e a t m e n t s g iv e n .

R e p e a te d a s s a y o f the sam e v i r u s p r e p a r a t io n on

u n t r a n s f o r m e d F B S C r e s u l t e d in no s ig n i f i c a n t d i f f e r e n c e s

in L o g j^ ^ T D 5 0 o v e r t h i r t y p a s s a g e s . P a i r e d t r a n s f o r m e d

F B S C c u l t u r e s re ta in e d t h e i r t r a n s f o r m e d c h a r a c t e r i s t i c s

f o r tw e n ty - s ix p a s s a g e s and then p r o g r e s s i v e l y l o s t t h e i r

t r a n s f o r m e d a p p e a ra n c e . A t te m p ts to re t r a n s f o r m these

r e v e r t a n t c u l t u r e s by B P V in fe c t io n w e r e u n s u c c e s s fu l .

1 2 6 .

%

FIGURE 20 L o w p o w e r p h o to m ic r o g r a p h s o f B P V - t r a n s f o r m e d

in d u c e d nude m o u s e t u m o u r s a) 16 d ays p o s t

in o c u la t io n b) 30 days p o s t in o c u la t io n .

S ta in e d w i t h M S B . B a r = 1 m m .

. 12 7 ,

0%

%

'g

F IG U R E 21 a) Pligh p o w e r p h o to m ic r o g r a p h o f in v i t r o

t r a n s f o r m e d c e l l s , f i x e d in s i t u . S ta in e d

w i t l i M S B . M a g n i f i c a t io n x 500.

b) H ig h p o w e r p h o to m ic r o g r a p h o f nude m o u s e

tu m o u r in d u c e d by t r a n s f o r m e d c e l l s .

S ta in e d w i t h M S B . M a g n i f i c a t io n x 500.

. 128 .

4.4 Dis eus sionC e l l c u l t u r e s t r a n s f o r m e d by o th e r D N A v i r u s e s

have been show n to be tu m o u r ig e n ic in nude m ic e . H u m a n

c e l l s a re t r a n s f o r m e d by c y to m e g a lo v i r u s (C u lp e t a l . , l 9 7 l ) ;

E p s t e i n - B a r r v i r u s - s p e c i f i c n u c le a r m e m b r a n e a n t ig e n

p o s i t i v e t u m o u r c e l l s f r o m n a s o p h a ry n g e a l c a r c in o m a in m a n

have been p a s s a g e d in nude m ic e ( V u i l l a u m e e t a l . , 1977).

A d e n o v i r u s - t r a n s f o r m e d c e l l l in e s p r o d u c e n o n - m e t a s t a s -

i s in g t u m o u r s w h ic h m a y show in v a s io n o f l o c a l t i s s u e s

( G a l l im o r e e t a l . , 1977). C e l ls t r a n s f o r m e d by o th e r

m e m b e r s o f the p ap o va v i r u s g ro u p have a ls o been r e p o r te d

to p ro d u c e t u m o u r s i n nude m ic e . SV 40 v i r u s t r a n s f o r m e d

m o u s e fo e ta l b r a in c e l l s (B e n d a e t a l . , 1977); h u m a n -m o u s e

s o m a t ic c e l l h y b r id s f r o m m o u s e p e r i t o n e a l m a c ro p h a g e s

and SV40 t r a n s f o r m e d h u m a n c e l l s (A d e n e t a l . , 1977);

SV40 t r a n s f o r m e d h u m a n c e l l s ( K o p r o w s k i and C ro c e , 1977);

p o ly o m a t r a n s f o r m e d m o u s e c e l l s (G ra d y e t a l . , 1977); and

M M V t r a n s f o r m e d nude m o u s e k id n e y c e l l s (C o s ta e t a l . , 1977)

have p ro d u c e d tu m o u r s w h e n in o c u la te d in to nude m ic e .

H o w e v e r , th e re a r e no r e p o r t s in the l i t e r a t u r e o f p a p i l l o m a

v i r u s t r a n s f o r m e d c e l l s p ro d u c in g t u m o u r s in nude m ic e .

The o n ly o th e r r e p o r t o f b ov ine tu m o u r c e l l i n d u c t io n o f

t u m o u r s in n o n - i r r a d i a t e d nude m ic e in v o lv e d the in o c u la t io n

o f c e l l s f r o m b o v in e o c u la r s q ua m o u s c a r c in o m a (H o f fm a n

e t a l . , 1977). The r e s u l t s r e p o r te d in t h i s p a p e r t h e r e f o r e

r e p r e s e n t the f i r s t t im e a p a p i l l o m a v i r u s t r a n s f o r m e d c e l l

c u l t u r e has in d u c e d tu m o u r s in nude m ic e .

The nude m o u s e is s u r p r i s i n g l y r e s i s t a n t to

t u m o u r g r o w th . A l l o g e n ic and x e n o g e n ic t u m o u r s g ro w b e t t e r

and m e ta s ta s ise m o r e r e a d i l y in the s t r a in o r s p e c ie s o f

o r i g i n than in the nude m o u s e . In fa c t , t u m o u r s in nudes m a y

a c tu a l l y r e g r e s s a f t e r t e m p o r a r y g r o w th ( P r e h n , 1976). A l l •

o f the a fo r e - m e n t io n e d nude m o u s e t u m o u r s in d u c e d by o th e r

papova v i r u s t r a n s f o r m e d c e l l s f a i l e d to m e t a s t a s is e . T he •

f in d in g s i n th is c h a p te r a re s i m i l a r .

. 1 2 9 ,

B P V has been r e p o r te d to cause t u m o u r s in

C 3 H /e B m ic e in v iv o , and to t r a n s f o r m c e l l s f r o m C 3 H /e B ,

C 57 / B L and B A B B / c m ic e in v i t r o ( T h o m a s e t a l . , 19 64),

No i n f o r m a t i o n is a v a i la b le in the l i t e r a t u r e as to the t im e

d e la y b e tw e e n B P V in je c t i o n and t u m o u r p r o d u c t io n in

C 3 H /e B m ic e . H o w e v e r , in the r e l a t i v e l y s h o r t t im e '

b e tw e e n B P V i n je c t i o n a nd d e a th in the w o r k r e p o r t e d h e re ,

no t u m o u r d e v e lo p m e n t w a s d e te c te d e i t h e r a t the s i te o f

i n o c u la t io n o r e ls e w h e r e in the nude m o u s e .

T h is c h a p te r r e p o r t s f o r the f i r s t t im e a B P V

t r a n s f o r m e d c e l l c u l t u r e w h ic h has r e v e r t e d to a n o r m a l

g r o w th p a t t e r n in d is t in g u is h a b le f r o m p a i r e d t r a n s f o r m a t i o n

s e n s i t i v e c o n t r o l c u l t u r e s . The r e v e r t a n t c u l t u r e w a s n o t

s e n s i t i v e to t r a n s f o r m a t i o n by B P V even th o u g h p a i r e d

c o n t r o l c u l t u r e s a t the sam e p assage n u m b e r w e r e .

R e v e r t a n t c u l t u r e s have been r e p o r t e d f o r S V 40 t r a n s f o r m e d

c e l l s o f h a m s te r , m o u s e and m o n k e y o r i g i n and a n u m b e r

a re r e s i s t a n t to SV40 r e - i n f e c t i o n (C u lp e t a l 1 97 1 ;

W i ls o n e t a l . , 1976; D ia m a n d o p o u lu s e t a l . , 1976). A v i r a l -

i n h ib i t i n g f a c t o r has been d e m o n s t r a te d in p o ly o m a

t r a n s f o r m e d c e l ls ( B e r e b b i e t a l . , 1 977) w h ic h depended on

c e l l c o n ta c t .

T h e a bse n ce o f B P V in u s e d c u l t u r e m e d iu m ,

t r a n s f o r m e d c e l l s and m o u s e tu m o u r s c o n f i r m s e a r l i e r

f in d in g s on the b e h a v io u r o f B P V as r e v ie w e d by O ls o n e t a l . ,

(1969}P r e v io u s e v id e n c e th a t t u m o u r in d u c t io n in nude

m ic e c o u ld be e nh a n ce d by p r e - t r e a t m e n t w i t h i r r a d i a t i o n

( P r e h n , 197 6 ) s u g g e s ts th a t f u r t h e r w o r k on B P V t r a n s f o r m e d

. 130,

c e l l t u m o u r p r o d u c t io n in i r r a d i a t e d nude m ic e m s iy p r o v id e

u s e fu l a d d i t io n a l i n f o r m a t i o n

4. 5 S u m m a r y

T h re e g ro u p s o f tw e n ty a th y m ic nude m ic e w e re

in o c u la te d s u b c u ta n e o u s ly - one w i t h f o e ta l b o v in e s k in c e l l s

(F B S C ) t r a n s f o r m e d in v i t r o by b o v in e p a p i l l o m a v i r u s (B P V ) ;

one w i t h a s u s p e n s io n o f B P V ; and the t h i r d w i t h

u n t r a n s f o r m e d F B S C . A l l t r a n s f o r m e d F B S C in o c u la te d

m ic e sh ow e d c e l l o u tg r o w th f r o m the i n o c u la t io n s i te .

T u m o u r s g re w r a p id l y a t f i r s t , p r o g r e s s i v e l y in v a d e d

a d ja c e n t t i s s u e s and m a tu r e d in to w e l l d i f f e r e n t i a t e d

f i b r o m a s . No e v id e n c e o f t u m o u r r e g r e s s io n w a s seen.

A t t e m p t s to v i s u a l i s e B P V in t u m o u r s by n e g a t iv e s ta in in g

and th in s e c t io n e x a m in a t io n u n d e r the e le c t r o n m ic r o s c o p e

w e re n o t s u c c e s s fu l . A t t e m p t s to g r o w t u m o u r c e l ls in v i t r o

and to s e r i a l l y t r a n s m i t t u m o u r s f a i l e d . P a r a l l e l in v i t r o

c u l t u r in g o f t r a n s f o r m e d F B S C show ed a p r o g r e s s i v e lo s s o f

t r a n s f o r m a t i o n c h a r a c t e r i s t i c s and r e v e r t a n t c u l t u r e s d id

n o t t r a n s f o r m a if te r r e in f e c t i o n w i t h B P V . P a i r e d

u n t r a n s f o r m e d F B S C c u l t u r e s , h o w e v e r , r e m a in e d s e n s i t i v e

to B P V in d u c e d t r a n s f o r m a t io n .

No t u m o u r s d e v e lo p e d in u n t r a n s f o r m e d F B S C and

B P V in o c u la te d g ro u p s , h o w e v e r the m e a n s u r v i v a l t im e o f

the B P V in o c u la te d g ro u p w a s s ig n i f i c a n t l y le s s than the

o th e r g ro u p s .

, 131 .

CHAPTER 5 IN VIVO EFFECTS OF BOVINE PAPILLOMA VIRUS - TEE

EFFECTS OF INTRACEREBRAL INOCULATION OF BOVINE

PAPILLOMA VIRUS AND BPV - TRANSFORMED CELLS IN

EXPERIMENTAL CALVES.

5=1 I n t r o d u c t i o n

Bovine i / a p i I I orna v i r u s e x t ra c te d from cutaneous

f ib r o p a p i l lo m a s has been shown to cause f i b r o b l a s t i c neoplasms

o f the meninges when in o c u la te d i n t r a c e r e b r a l l y (Gordon and

O lson, 1 9 6 8 ) 0 The i s o l a t i o n o f BPV from a l im e n ta ry p a p i l lo m a s

was re p o r te d by J a r r e t t e t a l» , 1978; and a d e ta i le d account o f

th e i s o l a t i o n and t ra n s m is s io n o f BPV e x t ra c te d from te a t

le s io n s appears i n c h a p te rs 2 and 3» Chapter 7 r e p o r ts t h a t

BPV e x t r a c te d from te a t r i c e g ra in le s io n s and t e a t p a p i l lo m a s

does not t ra n s fo rm f o e t a l bov ine c e l l s i n v i t r o , even though the

c e l l s used were t r a n s fo r m a t io n s e n s i t i v e to BPV e x t r a c te d from

a poo led sample o f a l im e n ta ry t r a c t le s io n s and s in g le case

sampled te a t f ib r o p a p i l lo m a s . Sera from c a t t l e i n f e c t e d w i th

BPV from r i c e g r a in le s io n s and te a t p a p i l lo m a s do no t i n h i b i t

i n v i t r o t r a n s fo rm a t io n by cutaneous f ib r o p a p i l lo m a d e r iv e d BPV

w h i le sera from c a t t l e w i t h f ib ro p a p i l lo m a s do (c h a p te r 8 ) . Some

ev idence p resen ted i n c h a p te r 2 in d ic a te s p h y s ic a l and b io che m ica l

d i f f e r e n c e s between BPV e x t ra c te d from v a r io u s s k in and t e a t

le s io n s .

T h is c h a p te r r e p o r ts experim en ts designed to d e te c t

d i f fe r e n c e s between samples o f t e a t , cutaneous and a l im e n ta ry

d e r iv e d BPV in t h e i r a b i l i t y t o produce tumours f o l lo w in g

i n t r a c e r e b r a l i n o c u la t i o n . The f i n d i n g t h a t BPV tra n s fo rm e d

f o e t a l bov ine s k in c e l l s were tu m ou r ige n ic i n nude mice

(c h a p te r 4 ) , bu t f a i l e d to produce tumours f o l l o w in g in t r a d e rm a l ,

subcutaneous o r submucosal in o c u la t io n i n ca lv e s (c h a p te r 5 )

made i t im p o r ta n t t h a t the t u m o u r ig e n ic i t y o f BPV t ra ns fo rm e d

c e l l s be te s te d i n t r a c e r e b r a l l y i n the b o v in e .

5 »2 M a te r ia ls and Methods

5 . 2 . 1 Exper im e n ta l c a lv e s .

S ix , one to tv/o month o ld d a i r y c ro ss c a lv e s were

132

randomly separa ted i n t o two groups o f th re e animals® One group

was in o c u la te d w i th c e l l s w h i le the o th e r w i th v i r u s . Each

c a l f was a n a e s th e t is e d u s in g p e n to th a l and m a in ta in e d under

ha lo thaneybxygen s u p p l ie d u s in g an endo trach e a l tu b e .

Two s i t e s on the f r o n t a l bone ju s t above th e ju n c t io n

o f th e r i g h t and l e f t o l f a c t o r y lobes w i t h the cerebrum were

p repared f o r s u rg e ry by shav ing the s k in and swabbing w i th Io d in e

m i t i s s o lu t i o n . F o l lo w in g a sm a ll 0 hem s k in i n c i s i o n , a 2mm

m e d u l la ry bone p i n was used to d r i l l a sm a ll h o le t o a l lo w

i n t r a c e r e b r a l i n o c u la t i o n . Two s i t e s , one on the i . e f t shou lde r

and the o th e r on the l e f t h in d le g were s i m i l a r l y p re pa re d .1 -----

These s i t e s each re c e iv e d te n 0 .1m l in t ra d e rm a l i n j e c t i o n s . The

l e f t cerebrum and sh ou ld e r s i t e s were in o c u la te d with, e i t h e r v i r u s

o r t ra n s fo rm e d c e l l s (see l a t e r ) w h i le the r i g h t cerebrum and h ind

le g s i t e s re c e iv e d e x t r a c t s o f normal s k in o r c o n t r o l c e l l

i n o c u la t io n s . A l l i n o c u la t io n s con ta ined s t e r i l i s e d , f i l t e r e d

a c t i v a te d c h a rc o a l as p r e v io u s ly desc r ibed (c h a p te r 3 and 4) to

a c t as a marker f o r subsequent s a m p lin g -

At f o r t n i g h t l y i n t e r v a l s f o r the f i r s t 20 weeks p o s t -

i n o c u la t io n a l l s i x ca lv e s were b le d to p ro v id e serum and b lood

f o r h a e m a to lo g ic a l e x a m in a t io n . A t the same t im e , one

in t r a d e rm a l i n o c u la t io n s i t e from t}ie shou ld e r and h in d le g was

b io p s y sampled under l o c a l a na e s th e s ia u s in g X y lo c a in e . Samples

were f i x e d i n 10^ fo rm o l s a l in e b e fo re be ing p rocessed f o r

f u r t h e r h i s t o p a th o lo g i c a l e x a m in a t io n . S e c t io n s were r o u t i n e l y

s ta in e d w i t h H aem atoxy lin and E os in and o th e r s p e c ia l s ta in s

when a p p r o p r ia te .

T h i r t y - f i v e weeks f o l lo w in g i n o c u la t io n , a l l s i x

ca lv e s were s a c r i f i c e d p r i o r to d e ta i le d pos t mortem

e x a m in a t io n . Samples f o r f u r t h e r h i s t o p a th o lo g i c a l exam ina t ion

were p rocessed as d e sc r ib e d above.

5*2.2 VirusThree v i r u s samples were used, one f o r each c a l f .

(1 ) C a l f 91 re c e iv e d a 10% w/v suspension o f cutaneous

f ib r o p a p i l lo m a s d e r iv e d from a s in g le f i e l d case. C o n tro l

s i t e s were in o c u la te d w i th a 10% w/v suspension o f norm al s k in

from the same a n im a l . (2 ) C a l f 82 re c e iv e d a 10% w/v

133 .

suspension o f cutaneous f ib r o p a p i l lo m a s which developed a long

s c a r i f i c a t i o n l i n e s in o c u la te d w i th BPV d e r iv e d from a pooled

sample o f a l im e n ta ry t r a c t l e s io n s . T h is sample was p ro v ide d

by P ro fe s s o r J a r r e t t . C o n t ro l s i t e s were in o c u la te d w i th a

10% w/v suspension o f normal bov ine oesophagus. (3 ) C a l f 84

re c e iv e d a 10% w/v suspension o f s in g le case d e r iv e d r i c e g ra in

le s io n s (sample RGB in c h a p te r 2, t a b le 2 1 ) . C o n t r o l s i t e s

were in o c u la te d w i th a 10?é w/v suspension o f normal bov ine t e a t .

5 . 2 . 3 = C e l ls

F o e ta l b ov ine s k in c e l l s were e s ta b l is h e d as d e sc r ibed

i n c h a p te r C e l ls were in f e c te d i n suspension w i t h each v i r u s-2

sample (10 o n - p la te d i l u t i o n ) . The a l im e n ta ry and cutaneous

f ib r o p a p i l lo m a - d e r iv e d B P V - inocu la ted c u l t u r e s showed the t y p i c a l

t ra n s fo rm a t io n - a s s o c ia te d changes desc r ibe d i n c h a p te r 7, bu t the

t e a t r i c e g r a in le s io n d e r iv e d BPV in o c u la te d c u l t u r e s d id n o t .

C e l ls were BPV in fo td .ed d u r in g t h e i r 4 th passage and were

ha rves te d between passages 12-14 f o r c a l f i n o c u la t i o n . P a r a l l e l

u n in fe c te d c u l t u r e s from th e same source were a ls o h a rve s te d

between passages 12-14 f o r c o n t r o l s i t e c a l f i n o c u la t i o n . C e l ls

were suspended u s in g t ryp s in /ve rse n e ^w a sh e d tw ic e w i t h PBS warmed

to 37°C, and f o i l o w i ng c e n t r i fu g a t io n , re s u s p e n d e d i n s u f f i c i e n t 7

PBS to c o n ta in 10 l i v e c e l l s per m l. Ca lves 45, 8 l and 8 3

re c e iv e d c e l l s i n f e c t e d w i t h BPV from cutaneous f ib r o p a p i l lo m a ,

t e a t r i c e g r a in le s io n s and a l im e n ta ry f ib r o p a p i l lo m a

r e s p e c t i v e ly .

5 . 3 R e s u lts

Tab le 26 summarises the e f f e c t o f i n t r a c e r e b r a l and

in t r a d e rm a l in o c u la t io n o f BPV and BPV t rans fo rm ed s k in c e l l s i n

e x p e r im e n ta l ca lves,.

, 134 -

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135.

Calves showed le s io n s a t the te n in t ra d e rm a l v i r u s

in o c u la te d s i t e s . F ib ro p a p i l lo m a s developed on the cutaneous

f i b r o p a p i l lo m a and the a l im e n ta ry t r a c t le s io n - d e r i v e d BPV

in o c u la te d c a lv e s (91 and 8 2 ) w h i le t y p i c a l r i c e g r a in le s io n s

r e s u l te d on the te a t r i c e g ra in le s io n - d e r i v e d BPV in o c u la te d

c a l f ( 8 4 ) . At p os t-m o r te m ,35 weeks post i n o c u la t i o n , ca lve s 91

and 8? showed e x te n s iv e f i b r o b l a s t i c meningomas occupy ing

a p p ro x im a te ly one t h i r d o f the i n t r a c r a n i a l l e f t c e re b ra l

hemisphere ( f i g u r e 22 a) and b) ) . No tumour development

occu rred in the te a t r i c e g r a in - d e r i v e d BPV in o c u la te d c a l f 84.

No le s io n s developed a t any c o n t r o l i n t r a c e r e b r a l o r

in t r a d e rm a l in o c u la t io n s i t e s .

F ig u re 22a) M ac ioscop ic appearance o f BPV-induced

meningoma. C a l f 91- Bar = 2cm.

b) M ic ro s c o p ic appearance o f BPV-induced meningoma,

C a l f 91. Bar = 1mm. S ta in e d w i th H & E.

. 1 3 6 ,

Calves 4 5 , 8'1 and 8.3 wh ich were in o c u la te d w i th

c o n t r o l and BPV t ra n s fo rm e d o r in f e c te d f o e t a l bov ine s k in c e l l s

d id no t deve lop le s io n s a t e i t h e r in t r a d e rm a l o r i n t r a c e r e b r a l

s i t e s . H a e m a to lo g ica l exam ina t ion produced unrem arkab le r e s u l t s

which appear i n Appendix C o f t h i s t h e s is . H i s t o l o g i c a l

e xam in a t io n o f b io p sy samples p ro v ide d r e s u l t s s i m i l a r to those

d e s c r ib e d i n Chapter 3»

Gross exam ina t ion o f the c r a n ia l c a v i t y o f ca lve s

and 8 2 showed s u b s t a n t ia l com pressive d e fo rm a t io n o f the l e f t

e thmoid t u r b i n a t e s . A normal dura mater covered e x te n s iv e ,

m u l t i p le smooth round and f i r m le s io n s , encapsu la ted by a

g ro s s ly deformed l e f t cerebrum and o l f a c t o r y lo b e . Tumours

were opaque and w h ite on i n c i s i o n showing a d i s t i n c t l y f ib r o u s

t e x t u r e . H i s t o l o g i c a l l y , the tumours showed o n ly r a re evidence

o f in v a s io n o f a d ja c e n t c e n t r a l nervous t i s s u e and were seen to

be c o n t in u o u s w i th the p ia m a te r . The tumours were composed o f

r e l a t i v e l y a v a s c u la r , w e l l d i f f e r e n t i a t e d f i b r o u s t i s s u e , s m a l le r

nodu les showing c e l i s wnich were more f i b r o b l a s t i c i n appearance

(F ig u re 22 b) ) . H i s t o l o g i c a l exam ina t io n o f i n t r a c e r e b r a l

i n o c u la t i o n s i t e s on the r i g h t cerebrum o f c a lv e s 91 and 8 2 as

wel3 as b o th s i t e s o f the o th e r c a lv e s showed th e presence o f

carbon surrounded by m inor sca r t i s s u e . No ev idence o f an

in f la m m a to ry response was seen i n any i n t r a c e r e b r a l i n o c u la t io n

s i t e in c lu d in g the meningiomas o f ca lve s 91 and 8 2 ,

5 . 4 DISCUSSION

The p ro d u c t io n o f meningiomas e x p e r im e n ta l ly i n ca lve s

has been re p o r te d p r e v io u s ly . Gordon and O lson ( I 9 6 8 ) used

twentyône week o ld c a lv e s and, f o l lo w in g i n t r a c e r e b r a l

i n o c u la t i o n , seventeen c a lv e s developed f i b r o b l a s t i c neoplasms.

L ik e o th e r p apovav iruses (C hapte r 1 ) , BPV produces i n t r a c r a n i a l

13 7 .

f i b r o b l a s t i c neoplasms in th e hamster (Rob l e t a l . , 1972).

This chapter reports two calves which were 4-8 weeks old at inoculation and also developed fibroblastic neoplasms. The macroscopic and microscopic appearance of the tumours were in agreement with those reported earlier.

Gordon and O lson (1968) a ls o re p o r te d th e t ra n s m is s io n

o f cutaneous f i b r o p a p i l lo m a d e r iv e d BPV t o th e m uzzle , tongue

and rumen o f c a lv e s . C a l f 82 re p o r te d here was in o c u la te d w i th

BPV e x t r a c te d from cutaneous f ib r o p a p i l lo m a s w h ich developed on

a c a l f in o c u la te d with BPV e x t ra c te d from a poo led sample o f

a l im e n ta ry t r a c t p a p i l lo m a s , f ib r o p a p i l lo m a s and f ib ro m a s ,

(This material was supplied by Professor Jarrett.) This BPV sample was indistinguishable from normal cutaneous fibropapilloma derived BPV in its ability to produce meningomas.

While teat rice grain lesions have been transmitted to the skin of calves (see Chapter 3), neither the naturally occurring lesions nor those transmitted experimentally show evidence of fibrous tissue proliferation. This absence of a mesenchymal response is confirmed by the absence of in vitro transformation of foetal bovine skin cultures as well as the absence of fibrous meningoma production following intracerebral inoculation,

BPV-transform ed c e l l c u l t u r e s a re tu m o u r ig e n ic i n nude

mice (C hap te r 4) and i n v i t r o BPV t ra n s fo rm a t io n can be

i n h i b i t e d by a n t i s e r a from f ib r o p a p i l lo m a b e a r in g c a t t l e (C hapte r

8 ) , A d s o r b t i o n o f such se ra w i th BPV t ra n s fo rm e d c e l l s ; and

f ib ro m a , f i b r o p a p i l lo m a and BPV induced meningoma t i s s u e m arked ly

reduces the t r a n s fo r m a t io n i n h i b i t i o n t i t r e (C hap te r 8 ) . The

in t r a d e r m a l , submucosal and subcutaneous in o c u la t i o n o f BPV

t ra n s fo rm e d c e l l s has been shown to in c re a s e a n im a l r e s is ta n c e

t o c h a l le n g e w i t h f ib r o p a p i l lo m a d e r iv e d BPV b u t n o t t e a t

p a p i l lo m a o r r i c e g r a in le s io n d e r iv e d BPV (C hap te r 3)= The

absence o f tumour development f o l lo w in g i n t r a c e r e b r a l i n o c u la t io n

o f t ra n s fo rm e d c e l l s may have been due to the r e l a t i v e l y low

number ( 2 x 10^ ) o f c e l l s i n the ino cu lum , o r h ig h a n t i g e n i c i t y

o f the c e l l s . Another e x p la n a t io n i s t h a t th e t ra n s fo rm e d c e l l s

a re r e l a t i v e l y non-oncogen ic i n th e b o v in e . C o n s id e ra b ly more

. 1 3 8 .

work w i l l be necessary u s in g l a r g e r numbers o f an im a ls b e fo re

these m a t te rs can be re s o lv e d .

5=5 SUMMARY

In t r a c e r e b r a l in o c u la t io n o f two c a lv e s w i th

f ib ro p a p i l l . o m a -d e r iv e d BPV r e s u l t e d i n the p ro d u c t io n o f f ib r o u s

meningomas. One o f these c a lv e s was in o c u la te d w i t h BPV from

f ib r o p a p i l lo m a s which appeared on a c a l f e x p e r im e n ta l ly in o c u la te d

w i th BPV i s o la t e d from a poo led sample o f a l im e n ta ry t r a c t le s io n s ,

Three c a lv e s in o c u la te d w i th BPV t ra ns fo rm e d and un tra n s fo rm ed

f o e t a l b ov ine s k in c e l l s , f a i l e d to produce le s io n s f o l lo w in g

in t ra d e rm a l o r i n t r a c e r e b r a l i n o c u la t i o n . One c a l f in o c u la te d

w i th t e a t r i c e g r a in le s io n d e r iv e d BPV responded w i t h t y p i c a l

r i c e g r a in le s io n s f o l l o w in g in t ra d e rm a l i n o c u la t i o n b u t f a i l e d

to produce any le s io n f o l l o w in g i n t r a c e r e b r a l i n o c u la t i o n .

These f in d in g s a re d iscussed in r e l a t i o n to o th e r i n v iv o and i n

v i t r o d i f f e r e n c e s between BPV is o la te d from m o rp h o lo g ic a l ly

d i f f e r e n t l e s io n s .

.139

C H A P T E R 6 : T H E E F F E C T O F P A S T E U R IS A T IO N O NB O V IN E P A P I L L O M A V IR U S I N V IV O A N D IN V IT R O .


In a r e c e n t s u r v e y (C h a p te r 2) 36% o f a l l c a t t le and

48% o f p a r o u s fe m a le c a t t le w e re a f fe c te d w i t h te a t

p a p i l l o m a t o s i s . The c o n s is te n t d e m o n s t r a t i o n o f b o v in e

p a p i l l o m a v i r u s (B P V ) i n n e g a t i v e ly s ta in e d p r e p a r a t io n s o f

p o o le d and i n d i v id u a l p a p i l l o m a s le d to the s u g g e s t io n th a t

m i l k f r o m a f fe c te d c a t t le c o u ld c o n ta in v i r u s r e le a s e d by

the s u p e r f i c i a l t r a u m a o f m a c h in e m i l k i n g ( C h a p te r 2).

B o th c a lv e s and m a n m a y t h e r e f o r e be e x p o s e d to a k n o w n

o n c o g e n ic b ov ine v i r u s w h e n fe d m i l k f r o m a f fe c te d c o w s .

T h is c h a p te r r e p o r t s the in v iv o and in v i t r o e f f e c t o f

p a t e u r i s a t i o n on d i f f e r e n t i s o la t e s o f b o v in e p a p i l l o m a v i r u s

e x t r a c t e d f r o m th r e e m o r p h o lo g i c a l l y s e p a ra b le te a t

p a p i l l o m a le s io n ty p e s a nd tvv o s k in f i b r o p a p i l l o m a ty p e s .

T h e p re s e n c e o f b o v in e p a p i l l o m a v i r u s p a r t i c l e s in tw o o f

s ix m i l k s a m p le s b ou g h t a t r e t a i l o u t le ts i s a ls o r e c o r d e d .

6 , 2 M a t e r i a l and M e th o d s

6 . 2 . 1 C la s s i f i c a t i o n o f L e s io n T yp es

E a c h v i r u s e x t r a c t w a s d e r i v e d f r o m a s in g le case

a f fe c te d w i t h o n ly one le s io n ty p e . T h e t h r e e te a t p a p i l l o m a

ty p e s have been p r e v io u s l y d e s c r ib e d a nd th e y w e r e

p r o v i s i o n a l l y c a l le d f r o n d (F ) , f l a t a nd ro u n d (F R ) and r i c e -

g r a in (R G ) le s io n s (C h a p te r 2 ). T w o c u ta n e o u s

f i b r o p a p i l l o m a s a re m o r p h o lo g i c a l l y a nd e p id e m io lo g ic a l l y

s e j^ a ra b le . T h e t y p i c a l c u ta n e o u s f i b r o p a p i l l o m a d e s c r ib e d

in the l i t e r a t u r e , in v o lv e s both e p i t h e l i a l and m e s e n c h y m a l

e le m e n ts a p p r o x im a t e l y e q u a l ly . The a n o g e n i ta l

f i b r o p a p i l l o m a is a c a u l i f l o w e r l i k e le s io n , p r i m a r i l y

i n v o lv in g m e s e n c h y m a l e le m e n ts w i t h s e c o n d a r y e p i t h e l i a l

. 140 .

ch a n g e s . F o r a f u l l e r h i s t o p a th o lo g ic a l d e s c r ip t i o n o f these

f iv e le s io n ty p e s see C h a p te r 3.

6 . 2 . 2 V i r u s E x t r a c t i o n

P a p i l l o m a s v /e re r e m o v e d f r o m c a t t le and s to r e d

s e p a r a te ly in P B S / G ly c e r o l a t 4 ° C . One p a p i l l o m a f r o m

each o f the f i v e ca ses w as s a m p le d and p la c e d in 1 0 % f o r m o l

s a l in e aind g lu la r a ld e h y d e f i x a t i v e f o r f u r t h e r h i s t o p a th o lo g ic a l

and e le c t r o n m ic r o s c o p i c (E M ) e x a m in a t io n . A f t e r

h i s t o l o g i c a l c o n f i r m a t i o n o f l e s io n type , p a p i l l o m a s w e re

h o m o g e n is e d u s in g a S i l v e r son b le n d e r in to a 10% w / v c ru d e

s u s p e n s io n in p ho sp ha te b u f fe re d s a l in e (P B S ) . T h is

s u s p e n s io n w a s c l a r i f i e d u s in g an SW 27 r o t o r a t 10, 000 r p m

f o r 20 m in u te s in a B e c k m a n L -250 u l t r a c e n t r i f u g e . The

r e s u l t i n g s u p e rn a ta n t w a s then la b e l le d and s to r e d a t -7 0 ^ C

u n t i l a n im a ls c o u ld be in fe c te d . A l i q u o t s o f each e x t r a c t

w e re f u r t h e r p u r i f i e d a c c o r d in g to m e th o d s d e s c r ib e d

e a r l i e r ( L a n c a s t e r e t a l . , 1976) bu t w i t h o u t the use o f

d e te r g e n t o r u l t r a s o u n d to a id v i r u s d is p e r s i o n .

6 . 2 . 3 E le c t r o n M ic r o s c o p e E x a m in a t io n

E a c h c ru d e s u s p e n s io n w a s negathely s ta in e d w i t h

p h o s p h o tu n g S t ic a c id ( P T A ) and e x a m in e d f o r the p re s e n c e

o f B P V . P u r i f i e d p r e p a r a t io n s w e re s i m i l a r l y e x a m in e d

and v i r u s p a r t i c l e d ia m e te r m e a s u r e m e n ts w e r e m a d e as

p r e v io u s l y d e s c r ib e d (C h a p te r 2).

6 . 2 . 4 P a s t e u r i s â t ! on

One m l o f each v i r u s s a m p le w a s p la c e d in a h o t

w a t e r ba th h e ld a t 6 0 °C . A f t e r the t e m p e r a t u r e o f the

s a m p le re a c h e d 6 0 °C , t h i r t y m in u te s w a s a l lo w e d to e la p s e

b e fo re the s a m p le w as r e m o v e d and a l lo w e d to c o o l in a

r e f r i g e r a t o r h e ld a t 4 ^ C .

. 1 4 1 .

6 . 2 . 5 T e m p e r a t u r e - T im e I n a c t i v a t io n

F iv e a l iq u o ts o f the c u ta n e o u s f i b r o p a p i l l o m a B P V

s a m p le w e r e h e ld a t 6 0 °C f o r 30, 60, 120, 180, 240 m in u te s

and s u b s e q u e n t ly a s s a y e d to d e te r m in e the L o g T D ^ ^ . S ix

a l iq u o ts o f the sam e s a m p le w e re t r e a te d f o r 30 m in u te s

a t 50, 60, 7 0, 7 5, 80, 85 C and s u b s e q u e n t ly a s s a y e d to

d e t e r m in e the L o g TD5Q. (See C h a p te r 7 f o r d e ta i l s o f the

m e t h o d o lo g y ) .

6 . 2 . 6 In V iv o T i t r a t i o n

S ix m a jo r s k in s i te s on each t h r e e m o n t h - o ld c a l f

w e r e s e le c te d . E a c h s i te w a s p r e p a r e d by c l ip p in g the

h a i r f r o m tw o 1 0 x 2 0 c m a re a s w i t h a t l e a s t 1 0 c m o f

u n d ip p e d h a i r c o v e r e d s k in l e f t b e tw e e n t h e tw o c l ip p e d

a r e a s .

E a c l i p a s te u r is e d and u n t r e a te d v i r u s is o la te w a s

d i lu te d in f i v e 10 f o ld s teps w i t h P B S . D r i e d , f i l t e r e d and

s t e r i l i s e d a c t i v a te d c h a r c o a l w a s added to e a ch d i l u t i o n a t '

the ra te o f 10 m g p e r m l . T e n 0 .1 m l i n t r a d e r m a l

i n je c t io n s w e r e m a d e f o r each d i l u t i o n s te p in to the

a p p r o p r ia t e d e s ig n a te d s i te . A p p e n d ix D s u m m a r i s e s the

s i te s in o c u la te d w i t h the d i f f e r e n t d i l u t io n s o f b o th

p a s te u r is e d and u n t r e a te d v i r u s i s o la t e s . F o r t y c o n t r o l

in je c t io n s o f P BS w i t h 1 0 m g / m l a c t i v a te d c h a r c o a l w e re

m a d e .

B io p s y s a m p le s w e r e ta k e n a t f o r t n i g h t l y i n t e r v a l s"4.f r o m 1 0 " v i r u s d i l u t i o n s i te s o f b o th p a s t e u r i s e d and

u n t r e a te d is o la te s as w e l l as c o n t r o l s i t e s . S k in th ic k n e s s

m e a s u r e m e n ts w e re m a d e o f each i n je c t i o n s i te a t the sam e

t im e and the p re s e n c e o r a bsence o f p a p i l l o m a w a s n o te d .

E a c h b io p s y s a m p le w a s d iv id e d in to th r e e p a r t s , tw o p a r ts

w e r e f i x e d in 1 0 % f o r m o l s a l in e and g lu t a r a ld e h y d e f i x a t i v e s

f o r h i s t o l o g i c a l and E M e x a m in a t io n . T he o th e r p a r t w a s o

f r o z e n a t -7 0 C f o r f u t u r e use e i t h e r in v i r u s e x t r a c t i o n o r

f o r c r y o s t a t s e c t io n s .

, 1 4 2 .

6 . 2 . 7 M i l k S a m p l e s

S ix s e p a r a te r e t a i l o u t l e t s w e r e v i s i t e d i n one w e e k

and the e qu ivE i len t o f f i v e l i t r e s o f m i l k w a s p u r c h a s e d .

C a r e was taken to e n s u r e tha t each f i v e l i t r e s a m p le

c o m p r i s e d m i l k f r o m the s a m e d a i r y and w a s l a b e l l e d w i t h

the s a m e e x p i r y deite o r b a tc h n u m b e r . S a m p l e s w e r e

l a b e l l e d A - F and h a n d le d s e p a r a t e l y t h r o u g h o u t the

e x p e r i m e n t .

E a c h m i l k s a m p le w a s d i l u t e d 1:4 w i t h d i s t i l l e d

w a t e r a n d c e n t r i f u g e d f o r 10 m i n u t e s a t 2, 000 r p m on a

JS 7 . 5 r o t o r . The s u p e r n a t a n t w a s r e m o v e d a nd the

p r e c i p i t a t e r e s u s p e n d e d in 1 0 m l o f PBS f o r e v e r y 600 m l

o f o r i g i n a l m i l k s a m p l e . T e s t tubes c o n t a i n i n g the r e s u s p

ended p r e c i p i t a t e w e r e a l l o w e d to s tand o v e r n i g h t a t 4 ^C .

The f o l l o w i n g m o r n i n g , the s u p e r n a t a n t w a s r e m o v e d and the

u p p e r m o s t o r e p i t h e l i a l c o m p o n e n t of the c e l l p e l l e t was

c a r e f u l l y r e suspen ded in 0 . 5 m l o f P B S . E p i t h e l i a l c e l l s

w e r e p o o le d f o r each s a m p le , w a s h e d t w i c e i n P B S , p e l l e t t e d

a t 2, 000 r p m f o r 10 m i n u t e s and re s u s p e n d e d i n 1 m l o f P B S .

T h u s e a c h 5 l i t r e m i l k s a m p le w a s p r o c e s s e d i n t o a s in g le

1 m l e p i t h e l i a l c e l l s u s p e n s io n i n P BS ,

T h e s e 1 m l s u s p e n s io n s w e r e then s u b j e c t e d to t h r e e

f r e e z e - t h a w c y c l e s ( C h a p t e r 2) b e fo r e b e in g e x a m i n e d

n e g a t i v e l y s t a i n e d u n d e r the E M f o r the p r e s e n c e o f B P V .

6. 3 R e s u l t s

T a b le 26 l i s t s the r e s u l t s o f p a s t e u r i s a t i o n on i n v i v o

i n f e c t i v i t y o f v i r u s as m e a s u r e d by the a p p e a r a n c e o f

p a p i l l o m a s and f i b r o m a s a t the s i t e s o f i n j e c t i o n . No

s i g n i f i c a n t d i f f e r e n c e s w e r e d e te c te d b e tw e e n the t i t r e s o f

p a s t e u r i s e d and u n t r e a t e d v i r u s i s o l a t e s .

S k in t h i c k n e s s m e a s u r e m e n t s d e t e c t e d the p r e s e n c e .

o f an i n t r a d e r m a l f i b r o m a a t i n j e c t i o n s i t e s 2 - 4 w e e k s '

b e f o r e a p a p i l l o m a a p p e a r e d i n a l l c a l v e s e x c e p t those

, 143 ,

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144.

i n o c u l a t e d w i t h T F and T R G . H o w e v e r , no s i g n i f i c a n t

d i f f e r e n c e s o c c u r r e d b e tw e e n m a x i m u m m e a n s k i n

t h i c k n e s s m e a s u r e m e n t s a t each d i l u t i o n s i te o f p a s t e u r i s e d

and u n t r e a t e d v i r u s i s o l a t e s .

W h e n one v i r u s i s o l a t e f r o m c u ta n e o u s f i b r o p a p i l l o

m a s w a s h e l d a t 60 C f o r v a r i o u s t i m e i n t e r v a l s , a t e n - f o l d

r e d u c t i o n in t r a n s f o r m a t i o n t i t r e o c c u r r e d o n l y a f t e r f o u r

h o u r s a t 60 C had e la p s e d . H o w e v e r , w h e n the same

i s o l a t e w a s h e ld f o r 3 0 m i n u t e s a t v a r i o u s t e m p e r a t u r e s , a l l

d e te c t a b le t r a n s f o r m i n g a c t i v i t y w as l o s t a f t e r 30 m i n u t e s

a t 8 0 ° C . F i g u r e 23 i l l u s t r a t e s these o b s e r v a t i o n s

g r a p h i c a l l y .

The h i s t o p a t h o l o g i c a l e x a m i n a t i o n o f b i o p s y s a m p l e s

a r e r e p o r t e d i n g r e a t e r d e t a i l e l s e w h e r e ( C h a p t e r 3),

H o w e v e r , e ach v i r u s i s o l a t e p r o d u c e d e x p e r i m e n t a l l e s i o n s

i d e n t i c a l to the o r i g i n a l l e s i o n s f r o m w h i c h the i s o l a t e w a s

f i r s t e x t r a c t e d . No d i f f e r e n c e s w e r e d e t e c t e d b e tw e e n

l e s i o n s p r o d u c e d by p a s t e u r i s e d o r u n t r e a t e d a l i q u o t s f r o m

the s am e v i r u s i s o l a t e . M e a n p a r t i c l e d i a m e t e r s as

m e a s u r e d on n e g a t i v e l y s t a i n e d p r e p a r a t i o n s have been

r e p o r t e d p r e v i o u s l y ( C h a p t e r 2) b u t a r e l i s t e d on A p p e n d i x

D f o r the c o n v e n ie n c e o f the r e a d e r .

W h e n f r e e z e “ tha w e p i t h e l i a l c e l l s u s p e n s io n s f r o m

m i l k s a m p l e s f r o m s i x s e p a r a t e d a i r i e s w e r e e x a m i n e d

u n d e r the E M u s in g n e g a t i v e s t a i n i n g , two s a m p l e s y i e l d e d

p a r t i c l e s i n d i s t i n g u i s h a b l e i n s ize and m o r p h o l o g y , f r o m

b ov ine p a p i l l o m a v i r u s .

6. 4 D is eus s io n

T a b le 12 i s a s u m m a r y o f the h e a t s e n s i t i v i t y o f o t h e r

m e m b e r s o f the p a p o v a v i r u s g r o u p ( C h a p t e r 1). A l t h o u g h

i t ca n be seen f r o m the above r e s u l t s t h a t B P V i s r e s i s t a n t

to h e a t i t h o ld s t h i s p r o p e r t y i n c o m m o n w i t h o t h e r papo~.

v i r u s e s . O n l y one v i r u s , equ in e cu ta n e o u s p a p i l l o m a t o s i s ,

. 1 4 5 .

50

0 — 0 T E M P E R A T U R E ( C )

60 70 80 90

oLO

QH

o

1 4 5Z 3

© — © T i m e ( H o u r s )

F I G U R E 23: The t i m e " t e m p e r a t u r e i n a c t i v a t i o n o f b ov ine p a p i l l o m a v i r u s as m e a s u r e d by i n v i t r o t r a n s f o r m a t i o n .

o B P V h e l d f o r 30 m i n u t e s at v a r y i n g t e m p e r a t u r e s .

© B P V h e ld a t 6 0 °C f o r v a r y i n g t i m e s ,

c a l c u l a t e d u s in g m e t h o d s jD r e v io u s l y d e s c r i b e d .

. 1 4 6 .

does n o t s u r v i v e p a s t e u r i s a t i o n , th a t is h e a t in g to 6 0 °C f o r

3 0 m i n u t e s .

The w o r k r e p o r t e d i n t h i s p a p e r m a y tend to u n d e r

e s t i m a t e the r e s i s t a n c e o f B P V in m i l k to h e a t i n a c t i v a t i o n .

.Samples te s te d in t h i s r e p o r t w e r e 10% w / v s u s p e n s io n s of

. i .1 k X\>h\ laf i i ing (13 P 3 ^ h a :a c tu a l j a p j l i o m a s in P B S . i 1 k g |Bf%n has a

r e d u c e d w a t e r a c t i v i t y due to the h i g h e r c o n te n t of n e u t r a l /

s u g a r s a nd so the h e a t r e q u i r e d f o r c o m p l e t e i n a c t i v a t i o n

m a y be h i g h e r than the e x p e r i m e n t s i n d i c a t e . A d d i t i o n a l l y ,

the s p e c i f i c g r a v i t y o f m i l k a p p r o x i m a t e s t h a t o f B P V and

so any f r e e B P V i n m i l k i s l i k e l y to be e v e n l y d i s t r i b u t e d

t h r o u g h o u t the s a m p le . W hen m i l k f r o m s e v e r a l c o w s ,

o r f r o m s e v e r a l h e r d s a r e p oo le d i n a b u l k c o n t a i n e r , i t i s

l i k e l y th a t the w h o le bulk, s a m p le w i l l c o n t a i n B P V even i f

o n l y one cow is a f f e c t e d .

T h e se f i n d i n g s s u g g e s t th a t n o t o n l y c a l v e s d i r e c t l y

s u c k l i n g a f f e c t e d c o w s , but a l s o c a l v e s be ing a r t i f i c i a l l y

s u c k le d u s in g p a s t e u r i s e d w h o le m i l k o r s k i m m i l k a r e

l i k e l y to be e x p o s e d to i n f e c t i o u s B P V . T h e s e o b s e r v a t i o n s

a r e s u p p o r t e d by the f a c t t h a t i n the sam e a r e a w h e r e 48%

o f c o w s had t e a t p a p i l l o m a t o s i s , 1 9 % o f a l l c a t t l e had

p a p i l l o m a s o f the u p p e r a l i m e n t a r y t r a c t . A s i m i l a r ra n g e

o f l e s i o n typ e s w e r e r e p o r t e d i n the u p p e r a l i m e n t a r y t r a c t

as on the te a ts o f c a t t l e ( C h a p t e r 2 and J a r r e t t e t a l . , 197 8).

S e v e r a l r e p o r t s have s u gg e s te d th a t B P V i s

t r a n s m i s s i b l e to m a n (S c h u l t z , 1908; F r e n z , 1941; G r e n i e r

e t a i . , 1 9 7 6 ) and t h i s i s s u | j p o r t e d by the o b s e r v a t i o n tha t

p e r soins in c e r t a i n o c c u p a t i o n s such as m e a t h a n d l e r s a r e

a t h ig h r i s k of d e v e lo p in g w a r t s ( P e r el and .L u m p k in , 197 6).

H o w e v e r , o t h e r a u t h o r s c o n c lu d e th a t t h i s i s n o t the case

(R o w s o n and M a h y , 1967). The s u g g e s t i o n t h a t d i f f e r e n t

b ov ine p a p i l l o m a v i r u s e s o c c u r on b o v in e te a ts ( C h a p t e r s

2, 3 and 5) m a k e s i t i m p o r t a n t to e v a lu a te the se v i r u s e s i n

r e l a t i o n to p a t h o l o g i c a l l y s i m i l a r o r a l l e s i o n s a l r e a d y

. 1 4 7 .

r e c o g n i s e d i n m a n . The n a t u r a l l y o c c u r r i n g b o v in e te a t

r i c e g r a i n l e s i o n y i e l d s v i r u s w h i c h on t r a n s m i s s i o n

p r o d u c e s i d e n t i c a l l e s i o n s on the s k in o f c a l v e s ( C h a p t e r 3) -

t h i s l e s i o n i s o f s i m i l a r a p p e a r a n c e to f o c a l e p i t h e l i a l

h y p e r p l a s i a ( P r a e t o r i u s “ C la u s e n , 197 2). i n m a n . The

n a t u r a l l y o c c u r r i n g b o v in e te a t f r o n d p a p i l l o m a y i e l d s v i r u s

w h i c h on t r a n s m i s s i o n p r o d u c e s i d e n t i c a l l e s i o n s on the s k in

o f c a l v e s ( C h a p t e r 3) - t h i s l e s i o n i s o f s i m i l a r a p p e a r a n c e

to v e r r u c a v u l g a r i s i n m a n ( P r a e t o r i u s - C l a u s e n , 1972).

B o v in e p a p i l ] o m a v i r u s can be r e a d i l y d e m o n s t r a t e d i n m i l k

on r e t a i l sa le f o r h u m a n c o n s u m p t i o n , so h u m a n s i n a r e a s

w i t h a h ig h p r e v a l e n c e o f b ov ine tea t p a p i l l o m a t o s i s a r e

be ing e x p o s e d to a c t i v e v i r u s i n p a s t e u r i s e d m i l k . F u r t h e r

w o r k i s n e c e s s a r y to i n v e s t i g a t e the p a p i l l o m a v i r u s e s of

h u m a n o r a l l e s i o n s i n r e l a t i o n to those c a u s in g te a t

p a p i l l o m a s i n c a t t l e .

6, 5 S u m m ^ i r y

P r e v i o u s r e p o r t s o f a 48% p r e v a l e n c e o f te a t

p a p i l l o m a t o s i s a m o n g cow s in S co t la n d , l e d to an

iz ives t ige i t i on o f the e f f e c t s o f p a s t e u r i s a t i o n on bov ine

p a p i l l o m a v i r u s i s o l a t e d f r o m th r e e d i f f e r e n t t e a t l e s i o n

t ype s and tw o s e p a r a b le c u ta n e o u s f i b r o p a p i l l o m a t y p e s .

No s i g n i f i c a n t d i f f e r e n c e s i n t r a n s f o r m a t i o n t i t r e s o c c u r r e d

b e tw e e n p a s t e u r i s e d (6 0 ° C f o r 30 m i n u t e s ) a nd u n t r e a t e d

v i r u s s u s p e n s io n s i n v i t r o . S i m i l a r l y , in v i v o t i t r a t i o n

y i e l d e d no s i g n i f i c a n t d i f f e r e n c e s in the n u m b e r and

d e v e l o p m e n t o f f i b r o m a s and p a p i l l o m a s b e tw e e n p a s t e u r i s e d

and u n t r e a t e d s a m p l e s .

A v i r u s s u s p e n s io n o f one f i b r o p a p i l l o m a type w a s

s u b j e c t e d to v a r y i n g t i m e - t e m p e r a t u r e t r e a t m e n t s . A

te n fo ld r e d u c t i o n i n t r a n s f o r m a t i o n t i t r e o c c u r r e d a f t e r 4

h o u r s a t 6 0 °C and a l l t r a n s f o r m a t i o n a c t i v i t y w a s l o s t a f t e r

30 m i n u t e s a t 8 0 ° C .

U 8 .

B o v i n e p a p i l l o m a v i r u s p a r t i c l e s w e r e d e te c te d i n

two o f s ix w h o le m i l k s a m p l e s on sa le to the p u b l i c p u r c h a s e d

f r o m r e t a i l o u t l e t s i n one w e e k . The i m p o r t a n c e o f the se

f i n d i n g s a r e d i s c u s s e d in r e l a t i o n to the h ig h i n c i d e n c e o f

p a p i l l o m a s in the u p p e r a l i m e n t a r y t r a c t o f c a t t l e . A

s i m i l a r i t y i n a p p e a r a n c e b e tw e e n l e s i o n s on the bo v ine te a t

and those r e p o r t e d in the h u m a n o r a l c a v i t y i s r e c o r d e d .

1 4 9 .

C H A P T E R 7 I N V I T R O T R A N S F O R M A T I O N B YB O V I N E P A P I L L O M A V IR U S .


B o v in e p a p i l l o m a v i r u s ( B P V ) i s a m e m b e r o f the

p a p i l l o m a v i r u s s u b g r o u p o f the P a p o v a v i r i d a e . B P V h a s

...Luuu c u L ' rOi.t i . m t n a a t h e o t h e r papilloma viruses I n t r a c r a n i a l i n j e c t i o n o f B P V p r o d u c e s m e n i n g i o m a s in

c a l v e s and f i b r o b l a s t i c i n t r a c r a n i a l n e o p l a s m s i n the

h a m s t e r ( G o r d o n and O ls o n , 1 968; R o b l e t a l . , 1972).

S ub cu tan e o u s i n j e c t i o n o f B P V p r o d u c e s f i b r o m a s i n

C 3 H / c B m i c e and h a m s t e r s ( F r i e d m a n e t a l . , 1963;

C h e v i l l e , 1966; R o b l and O ls o n , 1968), and i n t r a d e r m a l

i n j e c t i o n o f B P V i n the h o r s e induces a c o n n e c t i v e t i s s u e

t u m o u r s i m i l a r to equ in e s a r c o i d , n a t u r a l l y o c c u r r i n g

ca ses o f w h i c h have been show n to c o n t a in B P V v i r u s -

s p e c i f i c D N A se qu e nce s ( O l s o n and Cook , 1951; L a n c a s t e r

et a l . , 1977).

R e p o r t s d u r i n g the e a r l y l 9 6 0 ’ s d e s c r i b e

t r a n s f o r m a t i o n in v i t r o by B P V . F o e ta l bov ine

c o n j u n c t i v a and k i d n e y c e l l l i n e s as w e l l as s e c o n d a r y

c u l t u r e s o f f o e t a l bov ine h e a r t t r a n s f o r m e d a f t e r i n f e c t i o n

w i t h B P V ( B l a c k e t a l . , 1963). B P V a l s o t r a n s f o r m e d ,

e m b r y o n i c p r i m a r y c e l l c u l t u r e s f r o m C 3 H / e B , C 5 7 / B L

and B A L B / c nzice ( T h o m a s e t a l . , 1964) as w e l l as f o e t a l

bo v in e s k in p r i m a r y c u l t u r e s ( T h o m a s e t a l . , 1963). S ince

tha t t i m e , no f u r t h e r r e p o r t s c o n c e r n i n g the use o f B P V

in d u c e d t r a n s f o r m a t i o n as an a s s a y s y s t e m have been

p u b l i s h e d . U n l i k e o t h e r m e m b e r s o f the P a p o v a v i r i d a e ,

no r e l i a b l e in v i t r o m e th o d has been r e p o r t e d f o r the

g r o w t h o r the q u a n t i f i c a t i o n o f B P V . P h y s i c a l p a r t i c l e

c o u n t i n g u s in g the e l e c t r o n m i c r o s c o p e o r b i o c h e m i c a l

p r o t e i n e s t i m a t i o n s o f p a r t i c l e n u m b e r s b o th dep end on a

lo ng , p o ten ta 11 y d a m a g in g and w a s t e f u l p r o c e d u r e to

p u r i f y B P V f r o m i n v i v o le s i o n s . E v e n so, they g ive no

. 1 5 0

i n d i c a t i o n of the b i o l o g i c a l a c t i v i t y o f B P V in any g iv e n

p r e p a r a t i o n .

T h i s p a p e r r e p o r t s the s e a r c h f o r s u i t a b le f o e t a l

b ov ine c e l l c u l t u r e s f o r use in a t r a n s f o r m a t i o n a s s a y

and the d e v e l o p m e n t and c h a r a c t e r i s a t i o n o f a q u a n ta l

t r a n s f o r m a t i o n a s s a y f o r B P V .

7 . 2 M a t e r i a l s and M e t h o d s

7 . 2 . 1 P r i m a r y C e l l C u l t u r e s

T a b l e l i s t s the v a r i o u s t i s s u e s c o l l e c t e d f r o m

b ov ine f o e t u s e s a t d i f f e r i n g s t a g e s o f g e s t a t i o n as

m e a s u r e d by f o e t a l l e n g t h ( n u c h a l c r e s t to t a i l b u t t ) .

A p p r o x i m a t e l y 0 . 5 to 1 g r a m o f f o e t a l t i s s u e , c o l l e c t e d

u n d e r s t e r i l e c o n d i t i o n s , w a s r i n s e d t h r e e t i m e s i n B a s a l

E a g le s m e d i u m ( B M E ) . 10 m l o f B M E w a s added to the

t i s s u e and the m i x t u r e t r e a t e d in a S t o m a c h e r ( A . J . S te w a r d ,

B l a c k f r i a r s Road, L o n d o n ) f o r 1 - 5 m i n u t e s d ep e n d in g on

the type o f t i s s u e . The r e s u l t a n t c e l l s u s p e n s io n w a s then

e v e n l y d i v i d e d i n to f o u r 8oz g la s s m e d i c a l f l a s k s a l r e a d y

c o n t a i n i n g 2 0 m l B M E w i t h 20% added f o e t a l b ov ine s e r u m .

7 - 1 0 d ays a f t e r se e d in g , b o t t l e s w e r e e x a m i n e d f o r

e v id e n c e o f c e l l g r o w t h . T h o s e w h i c h show ed g r o w t h

w i t h o u t e v id e n c e o f c o n t a m i n a t i o n w e r e p a s s a g e d a t a 1:2

r a t i o . T h i s p r o c e d u r e w a s r e p e a t e d a t w e e k l y i n t e r v a l s

u s in g B M E w i t h 15% f o e t a l b ov ine s e r u m u n t i l the c u l t u r e s

s to p p e d g r o w i n g .

7 . 2 . 2 S k in C e l l s

T h r e e c o l l e c t i o n m e th o d s w e r e used to o b t a i n

m a t e r i a l i n a t t e m p t s to e s t a b l i s h f o e t a l b o v in e s k in c u l t u r e s .

(1) P i e c e s o f f o e t a l b ov ine s k in ( 3 - 5 m m d i a m e t e r ) w e r e

t r e a t e d in the S t o m a c h e r as d e s c r i b e d above ; (2) 2 -3 p i e c e s

of f o e t a l bov ine s k in w e r e p la c e d in 8oz m e d i c a l f l a s k s , .

e p i d e r m i s u p p e r m o s t , i n 20 m l B M E w i t h 20% a dde d f o e t a l

. 151 ,

bov ine se runz ; (3) an a r e a 4 c m x 2 crn o f the s k in o f a

b ov ine fo e tu s w a s l i g h t l y s c r a p e d w i t h a s t e r i l e s c a lp e l

b lade and the s c r a p i n g s w e r e p la c e d in an 8 oz m e d i c a l

f l a s k in 20 m l o f B M E w i t h 20% f o e t a l b o v ine s e r u m .

7 . 2 . 3 M e n i n g e a l C e l l s

The v a s c u l a r m e n in g e s (p ia m a t e r and

a r a c h n o i d e a ) w e r e s e p a r a t e d f r o m the f i b r o u s m e n in g e s

( d u r a m a t e r ) . B o th w e r e t r e a t e d in the S t o m a c h e r b e fo r e

se e d in g on to 8oz m e d i c a l f l a s k s . I n b o v in e f o e tu s e s of

l e n g t h 40 c m , the m e n in g e s w e r e u n a v o i d a b l y c o n t a m i n a t e d

w i t h a c o n s i d e r a b l e a m o u n t o f b r a i n t i s s u e . I n s m a l l e r

f o e t u s e s , i t w a s p o s s i b l e to s e p a r a te the d i f f e r e n t

c o m p o n e n t s .

7 . 2 . 4 V i r u s

A 10% w / v h o m o g e n a te o f p a p i l l o m a t i s s u e w as

m a d e in p h o s p h a te b u f f e r e d s a l i n e (P B S ) u s in g a S i l v e r son

( S i l v e r son M a c h i n e s L t d . , W a t e r s i d e , C h e s h a m , E n g la n d )

b l e n d e r . T h i s w a s c e n t r i f u g e d a t 2, OOOg f o r 20 m i n u t e s

a t 4*^C in a B e c k m a n L 2 - 65 u l t r a c e n t r i f u g e u s in g a

SW27 r o t o r . P u r i f i e d v i r u s w a s p r e p a r e d a c c o r d i n g to

m e t h o d s d e s c r i b e d e a r l i e r ( L a n c a s t e r e t a l . , 1976) bu t

w i t h o u t the use o f d e t e r g e n t o r u l t r a s o n i c d i s p e r s i o n o f

p e l l e t s .

7 . 2 . 5 I n f e c t i o n O f C e l l s In S u s p e n s io n

A c o n f l u e n t m o n o l a y e r of each s e c o n d a r y c e l l

c u l t u r e w a s t r e a t e d w i t h t r y p s i n / v e r sene and a f t e r

w a s h in g fhe s u s p e n s io n o f c e l l s i n B M E + 10% f o e t a l b o v in e

s e r u m , the c e l l s w e r e seeded onto e a c h w e l l o f a 24 x 1 c m

F a l c o n M u l t i w e l l ( B e c to n & D i c k i n s o n L t d . , B r i n d l a y 74,4

R u n c o r n , C h e s h i r e , U K ) p la te a t the r a te o f 3 - 5 x 1 0

c e l l s p e r w e l l i n 0 . 9 m l o f B M E w i t h 15% f o e t a l bo v in e

. 1 5 2 .

s e r u m . W i t h i n 10 m i n u t e s , 0 .1 m l o f v i r u s s u s p e n s io n

a t s e r i a l 10 - f o l d d i l u t i o n s w as i n o c u l a t e d i n t o e a ch w e l l .

E a c h d i l u t i o n o f v i r u s w a s i n o c u l a t e d i n t o 4 w e l l s , thus

the r e s u l t i n g m u l t i w e l l p la te c o n ta in e d 4 r e p l i c a t e 10 -

f o l d s e r i a l v i r u s d i l u t i o n s o f 10^, lO ^ , 10^ , 10^, 10^ p lu s

4 c o n t r o l w e l l s w h i c h w e r e i n o c u l a t e d w i t h .1 m l 10%

c r u d e s u s p e n s io n o f n o r m a l s k i n t a k e n f r o m the sam e

a n i m a l as the v i r u s p r e p a r a t i o n . F o r a s s a y p u r p o s e s ,

the L og j^ ^50% t r a n s f o r m i n g dose ( T D 5 0 ) d e t e r m i n e d u s in g

1 0 - f o l d s tep d i l u t i o n , w a s m o r e a c c u r a t e l y c o n f i r m e d

u s in g r e p l i c a t e t w o - f o l d d i l u t i o n s teps a r o u n d the

t r a n s f o r m a t i o n end p o i n t .

7 . 2 . 6 I n f e c t i o n o f C e l l s A s M o n o l a y e r

M u l t i w e l l p la te s w e r e seeded w i t h 3 - 5 x 1 0 ^ c e l l s

i n . 9 m l B M E + 15% f o e t a l b ov ine s e r u m p e r w e l l and

p la c e d in a h u m i d i f i e d i n c u b a t o r w i t h a 5% C O ^ i n a i r

a t m o s p h e r e a t 37 C o v e r n i g h t . The f o l l o w i n g m o r n i n g

0 .1 m l c o n t r o l and v i r u s c o n t a i n i n g s u s p e n s io n s (as

d e s c r i b e d above ) w e r e i n o c u l a t e d i n t o the w e l l s .

7 . 2 . 7 E x a m i n a t i o n O f P l a t e s

M e d i u m w a s r e m o v e d f r o m e a c h p la te , the c e l l s

f i x e d f o r 30 m i n u t e s i n m e t h a n o l , r i n s e d w i t h w a t e r and

then s ta i n e d f o r 20 m i n u t e s u s in g C i e m s a . P l a t e s w e r e

r i n s e d a g a in i n w a t e r and a l l o w e d to d r y u p e n d e d on t i s s u e

p a p e r . F o r each p la te g r o s s e x a m i n a t i o n ( f i g u r e 24) w a s

f o l l o w e d by e x a m i n a t i o n on a E e i t z i n v e r t e d l i g h t m i c r o s c o p e

7 , 2 . 8 C a l c u l a t i o n O f L o g T D 50

The K a r b e r m o d i f i c a t i o n o f the R e ed and M e u n c h

m e t h o d w a s used to d e t e r m i n e the end p o i n t t i t r a t i o n o f

v a r i o u s v i r u s p r e p a r a t i o n s . To e s t i m a t e the a c c u r a c y and

r e p r o d u c i b i l i t y o f t r a n s f o r m a t i o n , t h r e e c e l l c u l t u r e s w e r e

1 5 3

m S

W m M M :

H IMw m B . fiiilii#

FIGURE 24 B P V t r a n s f o r m a t i o n o f b o v in e s k i n c u l t u r e s ,

a. and b . l o w p o w e r and m a c r o s c o p i c a p p e a r a n c e

ol c o n t r o l c u l t u r e s , c . and d. l o w p o w e r and

m a c r o s c o p i c a p p e a r a n c e o f B P V t r a n s f o r m e d

c u l t u r e s . G i e m s a s ta i n . B a r = 2 c m .

. 154 .

i n f e c t e d w i t h 10 s e p a r a t e a l i q u o t s o f B P V m a d e f r o m (1)

a s i n g le case p a p i l l o m a p oo l ; (2) 10 s e p a r a t e p a p i l l o m a s

f r o m the s a m e a n i m a l ,

7 . 2 . 9 C h a r ac te r i s a t i o n O f T r a n s f o r m i ng C e l l C u l t u r e s

E a c h c e l l c u l t u r e w h i c h show ed t r a n s f e r m a t i o n

w a s a s s a y e d to d e t e r m i n e the o p t i m a l t i m e f o r e x a m i n a t i o n .

T e n m u l t i w e l l p l a t e s jalus c e l l s w e r e p r e p a r e d , i n f e c t e d

i n s u s p e n s io n w i t h s e r i a l B P V d i l u t i o n s and s t a i n e d a t 2,

4, 6, 8, 10, 12, 14, 16, 18 and 20 d ays p o s t i n f e c t i o n .

The c e l l s w e r e n o t p a s s a g e d , h o w e v e r the m e d i u m was

r e p l a c e d a t w e e k l y i n t e r v a l s .

7 . 2 . 1 0 A d s o r p t i o n T i m e

A f o e t a l b ov ine s k in c u l t u r e w a s u s e d to

d e t e r m i n e the a d s o r p t i o n t i m e f o r B P V bo th a f t e r

i n f e c t i o n in s u s p e n s io n and as a m o n o l a y e r .

F o l l o w i n g i n o c u l a t i o n w i t h B P V , the i x ie d iu m w a s

r e m o v e d a t i n t e r v a l s as show n on f i g u r e d ; the w e l l s w e r e

r i n s e d tw i c e w i t h PBS and then 1 m l o f f r e s h B M E + 15%

fo e ta l bov ine s e r u m p la c e d in t o each w e l l . The p la te s w e r e

e x a m i n e d a t the o jo t im a l t i m e f o r t r a n s f o r m a t i o n d e t e r m i n e d

e a r l i e r .

7 . 2 . 1 1 G e n e r a t i o n I n t e r v a l

P a i r e d t r a n s f o r m e d and c o n t r o l c u l t u r e s w e r e

e x a m in e d a t r e g u l a r p assag e i n t e r v a l s to c o m p a r e g r o w t h

c h a r a c t e r i s t i c s . A s u s p e n s io n o f c e l l s w a s m a d e u s in g

t r y p s i n / v e r s f n e , c e l l c l u m p s w e r e d i s a s s o c i a t e d by

v i g o r o u s p i p e t t i n g p r i o r to c o u n t in g i n a h a e m o c y t o m e t e r .

V i a b i l i t y w a s e s t i m a t e d u s in g the t r y p a n b lue e x c l u s i o n

m e t h o d . G e n e r a t i o n i n t e r v a l s d u r i n g the lo g phase of

g r o w t h w e r e calculated f r o m g r o w t h c u r v e s p l o t t e d f o r

each c u l t u r e . S a t u r a t i o n d e n s i t i e s w e r e a l s o d e t e r m i n e d

1 5 5 ,

f o r each cv i l t v i re .

7 . 2 . 12 E s t i m a t i o n O f C e l l L o n g e v i t y

P a i r e d u n i n f e c te d and t r a n s f o r m e d c e l l c u l t u r e s

w e r e p a s s a g e d a t w e e k l y i n t e r v a l s a t a r a t i o o f 1:2 u n t i l

c e l l g r o w t l i c e a s e d . F o u r r e p l i c a t e s o f each c u l t u r e w e r e

t r e a t e d i d e n t i c a l l y f o r t h i s w o r k .

7 . 2 . 1 3 A g a r S u s p e n s io n C u l t u r e

A t t e m p t s w e r e m a d e to g r o w t r a n s f o r m e d c e l l s

i n a g a r s u s p e n s io n c u l t u r e s u s in g the m e t h o d d e s c r i b e d

by M a c P h e r son and M o n t a g n i e r (1964 ) .

7 . 2 . 1 4 Hae m a g g l u t i n a t i o n

The h a e m a g g l u t i n a t i n g a c t i v i t y o f B P V

p r e p a r a t i o n s w a s d e t e r m i n e d u s in g m o u s e e r y t h r o c y t e s

a c c o r d i n g to thetecknique de s c r i b e d by F a v r e e t a l . ( 1974 ) ,

S ix r e p l i c a t e t i t r a t i o n s u s in g t w o - f o l d d i l u t i o n s tep s w e r e

u sed to d e t e r m i n e the L o g ^ ^ H A D 50.

7 . 3 R e s u l t s

T a b l e 27 show s f o e t a l l e n g th , the t i s s u e s c o l l e c t e d ,

c u l t u r e s w h i c h g r e w b eyo nd the t h i r d p a s s a g e and those

w h i c h sh ow ed t r a n s f o r m a t i o n a f t e r i n f e c t i o n w i t h B P V .

C e l l c u l t u r e s f r o m f o e t a l b o v in e p a la te , c o n j u n c t i v a and

v a s c u l a r m e n i n g e s w e r e c o n s i s t e n t l y t r a n s f o r m e d by B P V .

O n l y tw o c e l l c u l t u r e s f r o m f o e t a l b o v in e s k i n t r a n s f o r m e d .

T h e s e w e r e two c u l t u r e s e s t a b l i s h e d u s in g the e p i d e r m a l

s c r a p i n g m e t h o d - c u l t u r e s f r o m the s a m e f o e tu s e s

e s t a b l i s h e d u s in g w h o le s k in p i e c e s o r ' s t o m a c h e d ' s k i n

yncces w e r e m o r e f i b r o b l a s t i c in a p p e a r a n c e and d id n o t

t r a n s f o r m a f t e r i n f e c t i o n w i t h B P V . No o t h e r f o e t a l

b ov ine s k i n c u l t u r e s e s t a b l i s h e d by a ny o f the t h r e e m e t h o d s

t r a n s f o r m e d w i t h B P V . I t s h ou ld be n o te d t h a t the f o e tu s e s

f r o m w h i c h the t r a n s f o r m a t i o n s e n s i t i v e s k i n c u l t u r e s w e r e

. 1 5 6 ,

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i s o l a t e d w e r e both n e a r - t e r m , sh o w in g a p p r o x i m a t e l y

1 c m h a i r l e n g th .

The f e a t u r e s of t r a n s f o r m a t i o n w e r e s i m i l a r i n a l l

c u l t u r e s . C u l t u r e s f i r s t show ed m o r p h o l o g i c a l changes

b e tw ee n 3-5 cla.yspost i n f e c t i o n , w i t h the c o n c u r r e n t

a p p e a r a n c e o f ' f l o a t e r ' c e l l s i n the m e d i u m and lo n g

s p in d le shaped c e l l s s h o w in g d i s o r g a n i s e d g r o w t h p a t t e r n s

The s p in d le c e l l s o v e r g r e w the o t h e r c e l l s a nd f o r m e d

in t o l a r g e b u n d le s o r c o r d s of r a p i d l y g r o w i n g c e l l s .

A t the j u n c t i o n o f these c o r d s the s p in d le c e l l s c r o s s e d

o v e r each o t h e r to f o r m the t y p i c a l t r a n s f o r m a t i o n f o c i

a s s o c i a t e d w i t l i c e l l t r a n s f o r m a t i o n by o t h e r m e m b e r s o f

the p a p o v a - v i r us g r o u p . The m e d i u m i n i n f e c t e d w e l l s

s h ow ed c o n s i d e r a b l e a c i d i f i c a t i o n w h e n c o m p a r e d w i t h

c o n t r o l w e l l s . B e tw e e n days 5 - 7 th e s e c o r d s o f c e l l s

had o v e r g r o w n to the e x te n t tha t the y w e r e v i s i b l e

m a c r o S C O p ic a l l y a s w i s p s ( F i g u r e 27) .

C o m p a r i s o n b e tw e e n B P V i n f e c t i o n o f c e l l s i n

s u s p e n s io n and as a m o n o l a y e r c o n s i s t e n t l y sh ow ed the

a p p e a r a n c e o f w i s p s to be d e la y e d by 3 - 4 d ays i n the

m o n o l a y e r i n f e c t e d c u l t u r e s . The L o g T D 50 d id n o t

d i f f e r s i g n i f i c a n t l y b e tw e e n the two i n f e c t i o n m e t h o d s .

The d e la y in a p p e a r a n c e o f w i s p s c o u ld be a s c r i b e d to

d i f f e r e n c e s in a d s o r p t i o n t i m e { F i g u r e 25) .

F i g u r e 25 show s the t i m e n e c e s s a r y f o r c o m p l e t e

v i r u s a d s o r p t i o n to take p lace to p ro d u c e m a x i m u m

L o g T D 50. C o m p l e t e a d s o r p t i o n of B P V t a k e s 3 - 4 days

w hen c e l l s a r e i n f e c t e d in s u s p e n s io n and. up to 7 d ays

w h e n c e l l s a r e i n f e c t e d as a m o n o l a y e r .

R e p e a te d p a s s a g in g o f p a i r e d t r a n s f o r m e d and

c o n t r o l c u l t u r e s show ed a c o n s i s t e n t i n c r e a s e in the

l o n g e v i t y of t r a n s f o r m e d c u l t u r e s w h e n c o m p a r e d with,

c o n t r o l s . T a b le 28 shows these d i f f e r e n c e s f o r f o u r

c u l t u r e s thus f a r o b s e r v e d in th i s l a b o r a t o r y . I n each

, 1 5 8 .

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T A B L E 2 8 : L O N G E V I T Y O F P A I R E D T R A N S F O R M E D A N DC O N T R O L C U L T U R E S .

C U L T U R E S T R A N S F O R M E D C O N T R O L

T o t a l N o , P a s s a g e s

T i m e( m o n th s )

T o t a l No . P a s s a g e s

T im e ( m o n t h s )

2 M e n in g e s 32 8 9 23 C o n j u n c t i v a 20 5 8 25 M e n in g e s 27 6 11 3( 8 A ) M e n in g e s 25 6 8 2

f o u r r e p l i c a t e b o t t l e s u se d f o r each c e l l c u l t u r e .

T A B L E 2 9 : T H E R E S U L T O F T R A N S F O R M A T I O N A S S A Y O FT E N S E P A R A T E L Y P R O C E S S E D P A P I L L O M A S F R O M A S I N G L E C A S E (65819) O N T H R E E D I F F E R E N T F O E T A L B O V I N E C U L T U R E S .

T r a n s f o r m a t i o n A s s a y R e s u l t L o g j ^ T D 5 0 Ha e m a g g -

l u t i n a t i o n +

C e l l C u l t u r e 11 Sk in 12 Sk in12C o n j u n c t i v a

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d e t e r m i n e d u s in » 4 r e p l i c a t e 2 s tep s e r i a l t i t r a t i o n f o r each s a m p l e .

+ d e t e r m i n e d u s i n ^ 6 r e p l i c a t e 2 s tep s e r i a l t i t r a t i o n f o r each s a m p l e .

n . s . n o t s i g n i f i c a n t .

1 6 0 ,

case the m a c r o s c o p i c w i s p s o f the t r a n s f o r m e d c u l t u r e s

p e r s i s t e d t h r o u g h o u t t h e i r l i f e t i m e .

No s i g n i f i c a n t d i f f e r e n c e s o c c u r r e d b e tw e e n the

g e n e r a t i o n i n t e r v a l s o r s a t u r a t i o n d e n s i t i e s o f t r a n s f o r m e d

and c o n t r o l c u l t u r e s a f t e r i n o c u l a t i o n and t h r o u g h

to c e l l dea th , s e v e r a l m o n th s l a t e r . H o w e v e r , f i g u r e 26

i l l u s t r a t e s d i f f e r e n c e s i n g r o w t h r a t e a f t e r B P V i n f e c t i o n ,

w i t h an i n i t i a l d e p r e s s i o n (day 0 - 3) i n g r o w t h r a t e , a

l a r g e a m o u n t o f c e l l dea th , and then a p o t e n t i a t i o n o f c e l l

g r o w t h so th a t m o n o l a y e r s o b s e r v e d a t days 8 - 1 2 show ed

s i m i l a r c e l l n u m b e r s to c o n t r o l s .

T r a n s f o r m e d c e l l s d id n o t f o r m c o l o n i e s d u r i n g a

f o u r w e e k o b s e r v a t i o n p e r i o d a f t e r s u s p e n s io n i n 0 .3 %

a g a r . T h r e e d i f f e r e n t c e l l c u l t u r e s , e ach t r a n s f o r m e d by

d i f f e r e n t B P V i s o l a t e s , w e r e te s te d and i n no case was

c o lo n y f o r m a t i o n o b s e r v e d .

W h e n ten p a p i l l o m a s f r o m a s in g le case (65819)

w e r e s e p a r a t e l y p r o c e s s e d in t o i n d i v i d u a l 10% c r u d e

sus pen s io n s and then a s s a y e d on t h r e e d i f f e r e n t c e l l

c u l t u r e s , t h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s b e tw e en

the m e a n Hog TD 5 0 o f each c u l t u r e ( T a b le 29) u s in g

'S tu d e n t ' s t - t e s t ' . W h e n ten s e p a r a te a l i q u o t s o f a 10%

c r u d e s u s p e n s io n o f a n o t h e r case (66702) w e r e a s s a y e d on

the sa m e th r e e c e l l c u l t u r e s , no s i g n i f i c a n t d i f f e r e n c e s

w e r e f o u n d b e tw e e n the m e a n L o g T D 50 of e ach c u l t u r e

( T a b le 30) . A p p e n d i x E c o n ta in s the d e t a i l e d r e s u l t s of

w o r k r e p o r t e d h e r e . I t shou ld h o w e v e r be n o te d , th a t in

a l l cases the m e a n t i t r e s o f s e p a r a t e l y p r o c e s s e d

p a p i l l o m a s had a h i g h e r s t a n d a r d e r r o r w h e n c o m p a r e d

w i t h m e a n t i t r e s o f s e p a r a te a l i q u o t s o f a s in g le p o o le d

p a p i l l o m a s a m p le . In both e x p e r i m e n t s , the hlA t i t r e s

w e r e l o w e r than the t r a n s f o r m a t i o n t i t r e , and the s t a n d a r d

e r r o r s w e r e h i g h e r i n d i c a t i n g the l o w e r p r e c i s i o n o f the

H A a s s a y c o m p a r e d w i t h the t r a n s f o r m a t i o n a s s a y .

161 ,

70

60

510'10 121 6 82 43

t i m e ( d a y s )

F I G U I I E 2 6: E f f e c t of b ov i ne p a p i l l o m a v i r u s on g r o w t h c u r v e o f a f o e t a l

b o v i n e s k i n C u l t u r e .

o c u l t u r e i n f e c t e d w i t h B P V a t t i m e o i n s u s p e n s i o n

o p a i r e d c o n t r o l c u l t u r e i n f e c t e d w i t h n o r m a l s k i n e x t r a c t

a t t i m e o i n s u s p e n s i o n .

Y m e d i u m c h a n g e .

1 6 2 ,

D e t e r m i n a t i o n of the o p t i m a l t i m e f o r r e a d i n g a s s a y

p la te s show ed th a t t h e r e w as c o n s i d e r a b l e v a r i a t i o n

b e tw e e n c e l l c u l t u r e s . In e ach c e l l c u l t u r e t h e r e w a s a

2 d ay i n t e r v a l b e tw e e n the t i m e w h e n m i c r o s c o p i c

e v id e n c e f o r I I P V t r a n s f o r m a t i o n w a s p r e s e n t w i t h o u t

g r o s s wis]3 f o r m a t i o n and w h e n c u l t u r e s w e r e so o v e r -

c o n f l u e n t th a t g r o s s e x a m i n a t i o n f o r w i s p s w a s d i f f i c u l t .

D u r i n g t h i s Z day i n t e r v a l bo th m i c r o s c o p i c and

m t i c r o s cop ie e v id e n c e f o r t r a n s f o r m a t i o n w a s c l e a r l y

e v id e n t , and t h i s i s the p e r i o d u s e d to r e a d a s s a y p l a t e s .

T a b l e 31 i l l u s t r a t e s the v a r i a b i l i t y b e tw e e n c e l l c u l t u r e s ,

s h o w in g the t i m e o f f i r s t a p p e a r a n c e o f w i s p s i n m o n o l a y e r

i n f e c t e d as w e l l as s u s p e n s io n i n f e c t e d c u l t u r e s .

R P V e x t r a c t e d f r o m cu ta neo us f i b r o p a p i l l o m a ,

a n o g e n i t a l f i b r o p a p i l l o m a , a l i m e n t a r y f i b r o p a p i l l o m a and

t e a t f i b r o p a p i l i o m a o f c a t t l e have a l l i n d u c e d t r a n s f o r m a t i o n

i n the f o u r m o s t s t u d ie d c u l t u r e s (11 and 12 s k in ; 2

m e n in g e s and 12 c o n j u n c t i v a ) . V i r u s e x t r a c t e d f r o m

can ine skin p a p i l l o m a has no t i n d u c e d t r a n s f o r m a t i o n i n

any o f these f o u r c u l t u r e s , s i m i l a r l y , t e a t p a p i l l o m a and

r i c e g r a i n l e s i o n d e r i v e d B P V has n o t i n d u c e d t r a n s f o r m a t i o n

i n the a b o v e m e n t i o n e d f o u r c u l t u r e s .

7 . 4 D i s c u s s i o n

The r e s u l t s p r e s e n t e d in tab le 27 show t h a t c u l t u r e s

d e r i v e d f r o m f o e t a l b o v in e m e n in g e s , c o n j u n c t i v a , and

p a la te , c o n s i s t e n t l y t r a n s f o r m e d a f t e r i n f e c t i o n w i t h B P V .

B l a c k et a 1. , ( 1 963) fo u n d th a t B P V t r a n s f o r m e d b o v in e

k i d n e y and f o e t a l b o v in e c o n j u n c t i v a (D B G ) c e l l l i n e s , as

w e l l as s e c o n d a r y c u l t u r e s of f o e t a l bo v in e h e a r t . P r i m a r y

bov ine k i d n e y c u l t u r e s h o w e v e r , d id n o t t r a n s f o r m n o r d id

the two f o e t a l b ov ine k i d n e y c u l t u r e s r e p o r t e d i n t h i s p a p e r .

T h o m a s e t a l , , (1963 and 1964) r e p o r t e d B P V In d u c e d

t r a n s f o r m a t i o n o f f o e t a l bov ine s k i n c e l l s and p r i m a r y

, 1 6 3 ,

T A B L E 3 0 : T H E R E S U L T O F T R A N S F O R M A T I O N A S S A Y O FT E N A L I Q U O T S O F P O O L E D P A P I L L O M A S F R O M A S I N G L E C A S E (66702) ON T H R E E D I F F E R E N T F O E T A L B O V I N E C U L T U R E S .

T r a n s f o r m a t i o n A s s a y R e s u l t ' ’' L o g i ^ T C ^ ^

Ha.emagg - l u t i n a t i o n +

C e l l C u l t u r e 11 Sk in 12 S k in12C o n j u n c t i v a

L ° 8 l 0H A D 50

M e a n T i t r e 6 4 . 2 4 4 . 2 2 4. 21 2. 53S , E . 0 . 0 6 0. 07 0 .0 6 0 . 3 4

t - v a l v e

de g r ee s o f f re e do m s ig n i f i c a n c e

0.49

18n . s .

' W z c 0.45

1811 . s .

^ ' ' ^ 4 l S 0. 99

18 n. s.

* d e t e r m i n e d u s in g 4 r e p l i c a t e 2 s tep s e r i a l t i t r a t i o n o f ea ch a l i q u o t .

+ d e t e r m i n e d u s in g 6 r e p l i c a t e 2 s tep s e r i a l t i t r a t i o n o f each a l i q u o t ,

n. s n o t s i g n i f i c a n t

T A B L E 3 1 : T I M E O F F I R S T A P P E A R A N C E O F T R A N S F O R M A T I O N W ISPS I N S U S P E N S IO N A N D M O N O L A Y E R I N F E C T E D C U L T U R E S .

T i m e O f F i r s t A p p e a r a n c e O f W i s p s ( d a y s )

C E L L S U S P E N S IO N M O N O L A Y E RC U L T U R E I N F E C T E D I N F E C T E D

2 M e n in g e s 10 14

5 M e n in g e s 9 13

1 0 M e n in g e s 7 11

11 Sk in 8 12

12 S k in 8 12

12 C o n j u n c t i v a 10 14

, 1 6 4 ,

c u l t u r e s d e r i v e d f r o m f o e t a l b ov in e s k in p i e c e s . T h i s

c h a p t e r r e p o r t s o n l y two f o e t a l b o v ine s k i n c u l t u r e s w h i c h

t r a n s f o r m , and these w e r e d e r i v e d f r o m e p i d e r m a l

s c r a p i n g s o f two n e a r t e r m fo e t u s e s . P a r a l l e l c u l t u r e s

f r o m the s a m e fo e tu s e s , u s in g w h o le o r d i s p e r s e d s k in

p ie c e s , f a i l e d to t r a n s f o r m , as d id c u l t u r e s f r o m the s k in

o f y o u n g e r and s m a l l e r f o e t u s e s . A l t h o u g h B P V i n d u c e d

t u m o u r s a r c k n o w n to o c c u r in b ov ine m e n i n g e s ( G o r d o n &

O ls o n , 1 9 68) and p a la te ( J a r r e t t et a l . , 1978) i n v i v o ,

t r a n s f o r m a t i o n by B P V o f c u l t u r e s f r o m th e s e t i s s u e s in

the f o e tu s , l ias n o t been p r e v i o u s l y r e c o r d e d .

The m i c r o s c o p i c a i^p ea ra nce o f B P V i n d u c e d

t r a n s f o r m a t i o n has a l r e a d y been d e s c r i b e d ( B l a c k e t a l . ,

1963; T h o m a s c t a l . , 1963 & 1964) h o w e v e r the

d e v e l o p m e n t o f g r o s s w i s p s w he n t r a n s f o r m e d c u l t u r e s

r e a c h ove r c o n f l u e n c e , has no t been r e p o r t e d p r e v i o u s l y .

I t i s these w i s p s w h i c h p r o v i d e a s h a r p l y d e f i n e d end p o in t ,

m a k i n g a q u a n ta l t r a n s f o r m a t i o n a s s a y m o r e a t t r a c t i v e and

e asy to p e r f o r m , w h e n c o m p a r e d w i t h e x i s t i n g B P V a s s a y

s y s t e m s . I n o v e r 200 a s s a y s c o n d u c te d ,a p a r t l y

t r a n s f o r m e d w e l l has n e v e r been o b s e r v e d ; the p r e s e n c e o r

ab s e n c e o f w i s p s a p p e a r s to be an a l l o r n o th in g p h e n o m e n o n ,

I n c o n t r a s t , the p l e o m o r p h i c n a t u r e of s o m e c u l t u r e s

m a k e s e x a m i n a t i o n f o r m i c r o s c o p i c f o c i v e r y d i f f i c u l t , w i t h

the r e s u l t that: end p o in t s a r e o b s c u r e .

T r a n s f o r m e d c u l t u r e s showed a 2 . 5 - 3 - f o l d i n c r e a s e

in l i f e s p a n w i t h o u t l o s s o f m a c r o s c o p i c w i s p s , c o m p a r e d

w i t h p a i r e d c o n t r o l c u l t u r e s . E a r l i e r p a p e r s have r e p o r t e d

i n c r e a s e d l o n g e v i t y b e f o r e the c o m p l e t e l i f e span of

t r a n s f o r m e d c u l t u r e s had been d e t e r m i n e d ( T h o m a s e t a l . ,

1963 & 1964X

S u s p e n s io n i n f e c t e d c u l t u r e s c o n s i s t e n t l y show ed

t r a n s f o r m a t i o n w i s p s 3 - 4 days e a r l i e r th a n m o n o l a y e r

in f e c t e d c u l t u r e s i n the t o t a l t i m e r e q u i r e d f o r t r a n s f o r m

a t i o n w i s s to a p p e a r , T h e se i n t e r v a l s c o n t r a s t w i t h the

. 165 .

p e r i o d o f 4 5 d a y s r e p o r t e d by ( T h o m a s e t a l . , 1963) f o r

f o e t a l bov ine s k in , bu t a r e s i m i l a r to the 6 - lO days

seen in m o u s e c e l l s ( T h o m a s et a l . , 1964) and the 3 - 1 0

d ays o b s e r v e d w i t h b ov ine c o n j u n c t i v a ( D B G ) c e l l s . D B G

s u s p e n s io n i n f e c t e d c u l t u r e s t r a n s f o r m e d 7 d ays e a r l i e r

than th o s e in f e c t e d as a m o n o l a y e r ( B l a c k et a l . , 1963).

The d i f f e r e n c e b e tw e e n a d s o r p t i o n t i m e s o f the two

i n f e c t i o n m e t h o d s , p a r a l l e l s the d e la y i n a p p e a r a n c e o f

t r a n s f o r m a t i o n w i s p s in m o n o l a y e r i n f e c t e d c u l t u r e s .

P r e v i o u s r e p o r t s o f l o w t r a n s f o r m a t i o n t i t r e s m a y have

been due to i n c o m p l e t e B P V a d s o r p t i o n .

T r a n s f o r m e d c e l l s d id n o t f o r m c o l o n i e s i n a g a r

s u s p e n s io n , and t h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s in

the g e n e r a t i o n i n t e r v a l s o r s a t u r a t i o n d e n s i t y o f p a i r e d

t r a n s f o r m e d and c o n t r o l c u l t u r e s .

M e a n t i t r e s c a l c u l a t e d f r o m r e p e a t e d a s s a y s on t h r e e

d i f f e r e n t c u l t u r e s s h o w e d no s i g n i f i c a n t d i f f e r e n c e s . T h i s

o c c u r r e d bo th u s in g ten s e p a r a t e l y p r o c e s s e d p a p i l l o m a s

f r o m a s in g le c a s e , and ten a l i q u o t s o f a s in g le p a p i l l o m a

c a s e . T h e s e r e s u l t s i n d i c a t e t h a t the t r a n s f o r m a t i o n

a s s a y i s r e p r o d u c i b l e on c u l t u r e s d e r i v e d f r o m d i f f e r e n t

t i s s u e s o f the s a m e fo e t u s , and f r o m the s a m e t i s s u e s o f

d i f f e r e n t f o e t u s e s .

The use o f 1 c m d i a m e t e r m u l t i w e l l p l a t e s has m a d e

the a s s a y p r o c e d u r e bo th e a s i e r to p e r f o r m and has

a l l o w e d c o n s i d e r a b l e e c o n o m ie s i n the use o f c e l l s a n d

r e a g e n t s . F o u r s e p a r a t e m u l t i w e l l p l a t e s can be

p r e p a r e d u s in g one c o n f l u e n t 8oz m e d i c a l f l a s k o f p r i m a r y

c u l t u r e . 1 l a e m a g g l u t i n a t i o n t i t r e s i n t h i s w o r k v a r i e d

b e tw e e n 0. 5 and 2 log^ ^^d i lu t ions l e s s than e q u i v a l e n t

t r a n s f o r m a t i o n t i t r e s . T r a n s f o r m a t i o n t i t r e s in t h i s

l a b o r a t o r y have v a r i e d b e tw e e n 0 and 6 . 2 i n d i c a t i n g g r e a t e r

s e n s i t i v i t y c o m p a r e d w i t h the h a i e m a g g l u t i n a t i o n . The ■

c o m p a r a t i v e r e s u l t s i n d i c a t e th a t the t r a n s f o r m a t i o n a s s a y

, 1 6 6 ,

i s m e a s u r i n g a d i f f e r e n t p a r a m e t e r to the h a e m a g g l u t i n a t i o n

as say.

The e f f e c t s o f i n o c u l a t i o n o f B P V t r a n s f o r m e d c e l l

c u l t u r e s in to c a t t l e and nude m i c e has been r e p o r t e d i n

C h a p t e r s 3, 4 and 5 o f t h i s t h e s i s . O n l y t h r e e f i b r o -

p a p i l i o m a e x t r a c t s have n o t ca use d i n v i t r o t r a n s f o r m a t i o n ,

c o m p a r e d w i t h o v e r f i f t y w h i c h h a v e . T h e s e n o n

t r a n s f o r m i n g i s o l a t e s a r e d i s c u s s e d in g r e a t e r d e t a i l i n

C h a p t e r 8.

7 . 5 S u m m a r y

F o r t y - f o u r c u l t u r e s g r e w p a s t the t h i r d p as s a g e

f r o m t w e n t y d i f f e r e n t t i s s u e s s a m p le d f r o m t h i r t e e n b ov ine

f o e t u s e s o f d i f f e r i n g s i z e s . T w e n t y c u l t u r e s t r a n s f o r m e d

a f t e r i n f e c t i o n w i t h b o v in e p a p i l l o m a v i r u s . A l l f o e t a l

b ov ine p a la te , c o n j u n c t i v a and v a s c u l a r m e n i n g e a l c u l t u r e s

t r a n s f o r m e d h o w e v e r o n l y e p i d e r m a l s c r a p i n g o f s k i n f r o m

n e a r - t e r m f o e t u s e s y i e l d e d t r a n s f o r m a b l e s k i n c u l t u r e s .

T r a n s f o r m e d c u l t u r e s sh ow ed h e r i t a b l e m i c r o s c o p i c and

m a c r o s c o p i c chang es w h i c h p e r s i s t e d t h r o u g h o u t t h e i r

l i f e span . A l t h o u g h no d i f f e r e n c e s w e r e d e t e c t e d i n

g e n e r a t i o n i n t e r v a l o r s a t u r a t i o n d e n s i t y , t r a n s f o r m e d

c u l t u r e s s u r v i v e d 2 . 5 - 3 t i m e s as l o n g as p a i r e d c o n t r o l

c u l t u r e s . T r a n s f o r m e d c e l l s d id n o t p r o d u c e c o l o n i e s

w h e n g r o w n in a g a r s u s p e n s io n . U n l i k e p r e v i o u s l y

r e p o r t e d m i c r o s c o p i c f o c i , m a c r o s c o p i c w i s p s , f o r m e d

w h e n B P V i n f e c t e d c u l t u r e s r e a c h o v e r c o n f l u e n c e , p r o v i d e d

a c l e a r l y d e f i n e d end p o i n t f o r a q u a n ta l t r a n s f o r m a t i o n

a s s a y . No s i g n i f i c a n t d i f f e r e n c e s r e s u l t e d b e tw e e n m e a n

t i t r e s o f the sam e v i r a l e x t r a c t on tw o s k in c u l t u r e s o f

d i f f e r e n t f o e t u s e s o r a s k in and a c o n j u n c t i v a l c u l t u r e f r o m

the s a m e f o e tu s . B P V e x t r a c t s f r o m b o v in e tea t , s k in

and a l i m e n t a r y f i b r o p a p i l l o m a s t r a n s f o r m e d a l l f o u r

c u l t u r e s te s te d , h o w e v e r e x t r a c t s f r o m c a n in e s k in

p a p i l l o m a , t e a t p a p i l l o m a and r i c e g r a i n l e s i o n s d id n o t .

A c o n s i s t e n t d i f f e r e n c e o f 4 days o c c u r r e d i n bo th the

B P V a d s o r p t i o n t i m e and the t i m e ta k e n f o r t r a n s f o r m a t i o n

w i s p s to a p p e a r w hen c e l l s i n f e c t e d i n s u s p e n s io n w e r e

c o m p a r e d w i t h m o n o l a y e r i n f e c t e d c u l t u r e s . The use o f

1 c m d i a m e t e r m u l t i w e l l p la te s r e s u l t e d i n an e a s i e r and

m o r e e c o n o m i c a l a s s a y p r o c e d u r e . T h e q u a n ta l

t r a n s f o r m a t i o n a s s a y r e p o r t e d in t h i s c h a p t e r has a g r e a t e r

s e n s i t i v i t y and p r e c i s i o n but m a y be m e a s u r i n g a d i f f e r e n t

B P V p a r a m e t e r than B P V h a e m a g g l u t i n a t i o n .

. 1 6 8 ,

Chapter 8 I n h i b i t i o n o f i n v i t r o t rons foT 'mat ion by bov ine

pa p i 1 1 oma v l r u n

8 o 1 L i t ro d uc t n

F ib rcp ani i IfHTia d e r i ved Bl^V has been shown t o produce

tumours o f both e p i t h o l i a l and mesenchymal t i s s u e s i n a wide

range o f ;u ia tomica l l o c a t i o n s i n c a t t l e (Gordon and Olson, '1968;

Chapters 2, '5, 9 o f t h i s t h e s i s ) * I n v i t r o t r a n s f o r m a t i o n o f

f o e t a l bov:i TV- si. In , p a l a t e , c o n j u n c t i v a and meningeal c u l t u r e s

has been d es c r ib e d e a r l i e r (Chapter 7 and r e fe r e n c e s t h e r e i n ) *

A n t i - v i r a l a n t i b o d i e s have been found i n f i b r o p a p i l l omas

(S m i th ie s and Olson, 1961) u s in g immuno- f luorescence. They may

be q u a n t i t a t e d u s in g im m uno d i f fus ion ( K o i 1 r r e t a l * , 1974) and

h a e m a g g lu t in a t i o n i n h i b i t i o n (Fa^re e t a l . , 1974). No

c o r r e l a t i o n was found between f ib roma r e g re s s io n and a n t i - V

a n t ib o d y by B a r th o ld and Olson ( I 9 ? 4 a ) , The f ib rom a c e l l s o f

BPV induced f i b r o p a p i l l omas la c k V - a n t i gens d e te c td b le b.y IF

and EM (S m i th ie s and O lson, 1 96 l ; Ta juna c t a l . , 1 9 6 8 ; Robl and

Olson, 1 9 6 8 ) bu t possess a su r face a n t ig e n d e te c ted by i n d i r e c t

IF on l i v e u n f i x e d BPV induced f ib rom a c e l l s grown i n v i v o and

i n v i t r o ( B a r t h o ld and Olson, 19?4b), S e r i a l l y sampled

f i b r o p a p i l l omas show oon.-d de rab le h i s t o p a t h o l o g i c a l ev idence o f

a ma jo r r o l e p layed by CMI i n the r e g re s s io n o f a t l e a s t the

f ib ro m a component o f bovine f i b ro pa p i l l o m a s ( B a r t h o ld and Olson,

1974b; Chapter d o f t h i s t h e s i s ) .

The i n v e s t i g a t i o n re p o r te d i n t h i s ch a p te r was

m o t iva te d by the need t o show c o n v i n c i n g l y t h a t the BPV i n v i t r o

t r a n s f o r m a t i o n was indeed BPV s p e c i f i c . Fu r the rm ore , d es p i te

repea ted a t te m p ts u s in g a v a r i e t y o f techn iq ue s r e p o r te d i n the

l i t e r a t u r e ( K o l l e r e t a l . , 197^; Alme ida and G o f fe , 1 9 6 5 and

o th e rs - see Chaptei- 1 , 5 ) , double im m unod i f fus ion o f a v a r i e t y

o f v i r u s i s o l a t e s , a n t i g e n p re p a ra to r y tech n iq ues and sera f rom

c a t t l e wi t } , r e g re s s in g f i b r o p a p i 1 1 omas f a i l e d t o produce d i s t i n c t

p r e c i p t i n l i n c s „ Tti i r. chaptcu- rc’ poT'ts e xp e r im en ta l work on the

i n t i i b i t i r r of in v i t r o v i r a l t r a n s fo r m a t i o n .

, 1 6 9 .

8.2 Material s and Methods

8 . 2 . 1 * V i r u s , C e l i a , Sera and Com plem ent.

The i s o l a t i o n and g ro w th o f t r a n s f o r m a t i o n s e n s i t i v e

f o e t a l b o v in e c e l l c u l t u r e s has been d e s f ; r ib e d e a r l i e r (C h a p te r

7 ) . One s ta n d a rd 109o w /v v i r u s s u sp e n s io n was used th ro u g h o u t

t h i s work (66702 ~ see C h a p te r ? )» V i r u s was s t o r e d i n

a l i q u o t s a t -70'^C u n t i l u s e .

S era were c o l l e c t e d from a number o f e x p e r im e n ta l

c a lv e s , e x p e r im e n t a l l y i n o c u la t e d w i t h d i f f e r e n t v i r u s i s o l a t e s

(See C h a p te rs 5 and 5 ) • The s e ra were h e a t i n a c t i v a t e d a t

60°C f o r 30 m in u te s b e fo r e b e in g h e ld a t ~20°C u n t i l f u r t h e r use,

A s ta n d a rd so u rce o f complemmi. was u s e d . Two one

week o ld lam bs were e x s a n g u in a te d and serum a l i q u o t s o f 9ml

were s to r e d a t --70^Vj t i l l u s e .

8 . 2 . 2 Assay P ro ce d u re

2 1 w e l l I 'talcon m u l t i w e l l t i s s u e c u l t u r e p la t e s were

u s e d . F ig u r e 2? i l l u s t r a t e s a t y p i c a l t e s t p l a t e . W e l ls A l

and B1 r e c e iv e d c o n t r o l c e l l s a l otm- and c o n tx 'o l c e l l s p lu s

a n t i serum p lu s complement r e s p e c t i v e l y , W e l ls C l and D1

r e c e iv e d c e l l s p lu s v i r u s p lu s complement and c e l l s p lu s v i r u s

respectively. A l l v ;e l1s i n row s 2 -6 i n c l u s i v e l y r e c e iv e d c e l l s

p lu s v i r u s a t 10 ^ d i l u t i o n ( e q u iv a le n t t o a p p r o x im a te ly 100

t r a n s f o r m in g u n i t s per w e l l ) p lu s com p lem ent. T e s t s e ra were

d i l u t e d t e n - f o l d s te p s u s in g E a g le s medium w i t h o u t added f o e t a l" -1b o v in e serum . Rows 2 , 3 , 4 , 5 and 6 r e c e iv e d u n d i l u t e d , 10 ,

-2 -3 -d10 , 10 and 10 d i l u t i o n s o f t e s t s e r a . Complement was

added as i n d i c a t e d above t o an on p l a t e c o n c e n t r a t i o n o f 3% by

vo lu m e . Each 1ml wêll t h u s c o n ta in e d - 0 .8 m l c e l l s , medium w i t h

19/0 added FBS, com plem ent; 0 .1 m l t e s t serum a t a p p r o p r ia t e-3

d i l u t i o n s ; and o . l m l o f v i r u s a t f i x e d d i l u t i o n o f 10

P la t e s were p la c e d i n a 3% 0 0 ^ , h u m i d i f i e d i n c u b a t o r

h e ld a t 3 7 °C . T h ree days l a t e r , th e medium was removed fro m

each w e l l and r e p la c e d w i t h 1 ml BME w i t h '\3% added FBS* P la t e s

were s t a in e d w i t h Giemsa and examined f o r e v id e n c e o f t r a n s

f o r m a t io n (see C h a p te r 7 ) . The 9 0 / t r a n s f o r m a t i o n i n h i b i t i o n

dose (Log^Q T ID 90) was c a l c u la t e d u s in g th e method o f Reed and

MuCnch.

.170.

% ■:

F ig u r e 2? BPV i n v i t r o t r a n s f o r m a t i o n i n h i b i t i o n assay t e s t

p l a t e . C o n te n ts o f w e l l s w e re ; A1 - c e l l s a lo n e ;

B1 - c e l l s p lu s a n t i serum p lu s com plem ent; C1 -

c e l l s , p lu s v i r u s p lu s com plem ent; D1 - c e l l s p lu s

v i r u s ; Row ? , 3 , k , 9 and 6 r e c e iv e d nea t 10 \

10 10 and 10 * d i l u t i o n s o f a n t is e r u m , 3%

complement and 10 BPV r e s p e c t i v e l y . The Log10

TIÜ90 o f t h i s serum (68888 Wl8) was 2 . 5 . S ta in e d

w i t h C iem sa. Bar = 1cm.

. 1 7 1 ,

8 , 2 , 3 V a r y in g complument and v i r u s conc e n t r a t i o n s

P r e l i m i n a r y e x p e r im e n ts had shown t h a t th e t r a n s

f o r m a t io n i n h i b i t o r y ( T I ) e f f e c t was a b se n t when b o th th e t e s t

s e ra and FBS i n th e medium were h e a t i n a c t i v a t e d b e fo r e u s e .

The T I e f f e c t c o u ld be r e s t o r e d u s in g an a l t e r n a t i v e so u rce o f

complement ( i n i t i a l l y g u in e a p ig serum, b u t s u b s e q u e n t ly

newborn lamb s e ru m ) *

V a ry in g c o n c e n t r a t io n s o f v i i ’us were used w i t h

s ta n d a rd c o n c e n t r a t io n s o f complement and t e s t serum, t o

i n v e s t i g a t e t h e l i - e f f e c t on th e serum T ID 5 0 , A d d i t i o n a l l y ,

v a r y in g c o n c e n t r a t io n s o f complement were used w i t h s ta n d a rd

c o n e p ' /T ra t i ons o f v i r u s and t e s t serum t o i n v e s t i g a t e t h e i r

e f f e c t on th e serum TJD 50

8 , 2 Al The e f f e c t o f h ig h t i t r e se ra on p r ev i o u s l y

t r a n s fo r m e d c e l l c u l t u r e s

A c e l l c u l t u r e t r a n s fo r m e d w i t h BPV (66702 ) was used

t o i n v e s t i g a t e w h e th e r h ig h T ID 50 s e ra i n h i b i t e d th e

p h e n o ty p ic e x p re s s io n o f t r a n s f o r m a t i o n r e p o r t e d e a r l i e r

(C h a p te r 7 ) - T e s t p ro c e d u re was as d e s c r ib e d e a r l i e r ( 8 , 2 , 2 )

w i t h t h e d i f f e r e n c e t h a t th e c o n t r o l w e l l s 1A , 1B, 1C and ID

a l l c o îd a in e d t ra n s fo r m e d c e l l s .

8 , 2 , 5 Adsorbtion of high titre sera with variou.s BPV

r e lated s uspensions.H igh t i t r e serum 68893 week 34 was a d s o rb e d w i t h an

e q u a l volume o f v a r io u s B P V - re la te d s u s p e n s io n s l i s t e d i n t a b l e

32, F o l lo w i r p ; thorough a g i t a t i o n , th e m ix tu r e s were

p la c e d i n a w a te r b a th h e ld a t 37°C f o r 30 m in u te s b e fo r e b e in g

h e ld a t 4 °c o v e r n i g h t , The f o l l o w i n g m o rn in g th e m ix tu r e s

were c e n t r i f u g e d f o r 30 m in u te s a t 2 ,0 0 0 rpm on a bench to p

,172,

TABLE 3B The e f f e c t on t r a n s f o r m a t io n i n h i b i t i o n t i t r e o£

| ) ] ' i o r serum a d s o r b t io n w i t h v a r io u s BPV r e l a t e d

su s p e n s io n s (serum 68888 W2h)

(■ th e se a re s i g n i f i c a n t l y (P <. 0 .0 1 ) d i f f e r e n t

from th e o t h e r t i t r e s )

Ads o r t a n t Log^Q TID 50

w(HOU-Poo

r-l

O

none 3-5n o rm a l b o v in e s k in 5-3norm al fo- l .a l t i fc su e 5.0n o rm a l f o e t a l t i s s u e c u l t u r ec e l ' s (10%) 5.25

t e a t f i b r o p a p i l l o m a 3.5t e a t r i c e g r a in l e s i o n 3.5dog s k in p a p i l lo m a 5.5t e a t p a p i l lo m a 5.5anogen i t a l f i b r o p a p i l l o m a 5.25cu ta n e o u s f i b r o p a p i l l o m a 1.75*

t e a t f i b r o p a p i l l o m a 3.5t e a t r i c e g r a in l e s i o n 5.75dog s k in p a p i l lo m a 3.5t e a t p a p i l lo m a 5.5anogeni t a l f i b r o p a p i l l o m a 5.0cu ta n e o u s f i b r o p a p i l l o m a 5.25

cu ta n e o u s f i b r o p a p i l l o m a 1.5*cu ta n e o u s f ib io m a 1.75*

7BPV t r a n s fo rm e d c e l l s (10 ) 1 .0*BPV meningoma 1.0*t e a t p a p i l l o m a 5.0r i c e g r a in 1er io n 3.5

rO

■HW

T3 aO O

(0H

omU}•H

+•>

. 173 .

c e n t r i f u g e .

Each s u p e rn a ta n t was th e n decan te d and t e s t e d t o

de te rm im -' i he T ID DO as d e s c r ib e d above ( 8 . 2 . 2 ) . To

compensate f o r the 50 d i l u t i o r i o f th e sej-a d u r in g a d s o r b t io n ,

0 .2 m l in s te a d o f 0 .1 m l was added pe r t e s t w e l l .

8 . 2 . 6 I n v e s t i g a t j on o f e f f e e t o f n o n - t r a n s fo r m in g i s o l a t e s

C h a p te r 7 r e p o r t s t h a t t h r e e o f o v e r $0 f i b r o p a p i l l o m a

i s o l a t e s f a i l e d t o t r a n s fo r m c u l t u r e s i n v i t r o . N e v e r th e le s s ,

th e s e i s o l a t e s were shown t o in d u c e m o r p h o lo g ic a l changes i n

v i t r o w h ic h were r e p r o d u c ib le and t i t r e d o u t t o lo w t i t r e s o f

be tw een 10 and 10 One o f th e s e i s o l a t e s was

in c lu d e d l)oth. i n th e a d s o r b t io r ' e x p e r im e n ts o u t l i n e d above

( 8 , 2 , 5 ) * I n a d d i t i o n , i s o l a t e s o f dog p a p i l l o m a , t e a t

p a p i l l o m a and r ic .> g r a in l e s i o n s were in c lu d e d i n th e

a d s o r b t i on expt-j im en l.f . ,

A two s ta g e assay was deve loped t o d e t e c t any

a d d i t i v e o r i n t e r f e r e n c e e f f e c t s o f n o n t r a n s fo r m in g v i r u s

i s o l a t e s on th e t i t r e o f t r a n s fo r m in g i s o l a t e s . I n t h i s

i n t e r f e r e n c e assay t'ne f i r s t s ta g e in v o lv e d p r e p a r in g a

t y p i c a l t r a n s f o r m a t i o n assay (d e s c r ib e d i n s e c t i o n 7 -2 ) u s in g

a n o n t r a n s fo r m in g i s o l a t e . F ou r days l a t e r , c e l l s on each

w e l l were suspended w i t h t r y p s i n / v e r s f o i e and d i v id e d o v e r two

f r e s h m u l t i w e l l d is h e s - each w e l l w h ic l i had r e c e iv e d a

p a r t i c u l a r n o n t r a n s fo r m in g v i r u s d i l u t i o n r e t a i n i n g i t s

p o s i t i o r on th e new p l a t e s . B o th new p l a t e s were th e n

i n o c u la t e d w i t h a t r a n s f o r m in g v i r u s i s o l a t e o f known t i t r e

i n a manner id f-u it i c a l t o a s ta n d a rd t r a n s f o r m a t i o n assay ( 7 * 2 ) ,

e xce p t t h a t eoch t r e a t e d w e l l r e c e iv e d th e e q u i v a le n t o f te n

t r a n s f o r m in g u n i t s o f th e second v i r u s . F u r t h e r e x p e r im e n ts

in v o lv e d th e s i m i l ta n eons i n f ec 1 ;i on o f c e l l s w i t h b o th

t r a n s f o r m in g and n o n t r a n s fo r m in g i s o l a t e s w i t h and w i t h o u t

th e a d d i t i o n o f 5% com p lem en t.

. 1 7 5 .

s

2

t_.o

TIMK ror iT jivOcuLA'nr»;. (WKKKS )

F ig u r e ?S : T r a i i r i 'o i - ’n a t io n In l r i V i t i oïi 11 t r e e of

exper ' i r . r n t a l c a l f ae ra in m l a t i (ut t o r o o t i n o c u la t 1 on

timefifdevc^l op!T;ont and rcgro ;- .r , ion o f f i b r o y ia p i l 1 omas3.

f i r s tappearance

o f f 1 b ro p.a 1 )i 11 0 '.a.a :

IK

f i r s t .h i s t o l o g i c a l ev i dence

O r c o l 1 d i a t: t d

inuî iune ro f j r c '» : ; " -

( i f a p p l i c a b le )

r e j e c t i o n o f f .i b r o p a p i l l omas com piete

. 1 7 6 ,

8 . i R p su 11 sFigure R8 illustrâtes the TID 50 titres of sera from

cattle expo)imentally infected with various BPV isolates. Full details of the transmission data of these calves appeals in Chapter 5 of this thesis. Calves which developed multiple fibropapillomas all showed detectable transformation inhibitory (TI) activity in tliei r sera. This TI activity was first detected in sera at the time of first appearance of masses of lymphocytes in the fibroma component indicating the onset of cell mediated regression of lesions. Calves 68891 and 68886 both successfully rejected their fibropapillomas and TI titres fell to undetectable levels 4-8 weeks following complete rejection. Calves 6889b and 68888 mounted a rejection response but failed to reject fibropapi11omas successfully and TI titres continued to show a progressive increase until post-mortem at 24 weeks. Serum sampl es from crilves i r.ocu': ated with transformed cells (49, 8 I, 8b, TI'BSC(i), TFBMC), control cells (FBSC, FBMC) and teat rice grain virus (68882, 84) failed to show detectable TI titres. Calves 82 and 68882 both showed small fibropapilloma lesions which were removed for biopsy early, in their development. For a more complete description of the transmission experiments refer to Chapters '5 and 5- Calves 6 8 8 9 1 and 8 2 both showed meningomas at post-mortem 55 weeks post inoculation. Histological examination failed to show evidence of cell mediated rejection, TI assay of serum samples taken at PM failed to show detectable TI activity. All titres shown on figure 2 8 are the means of four replicant assays.

Figure 29 shows the effect of varying virus and complement concentrations on the TID 50 of one serum sample diluted precisely to the previously determined TID 50 using 5%

o f ,complement and 100 transforming units^ner well. Each point on the graph represents the result of twelve replicate titrations. At very high concentrations of BPV the TI effect of the serum is reduced, however, over a wide range (2.5 log units) of concentrations, virus dilution does not alter the TI of the serum. When insufficient virus is present to transform the

,177.

Concen t r -n i i on o f v i " u ; ( t T'on.--forini ng u r . i t .o /m l )

K1C) 1 0

100

a)GUo

w

rHI-)C)

O * “” 0 Conc-Gnt ra t . 1 on o f O.oinp ! r. 'ni 't;1' (à' I y ' v o l u m e )

F i g u r e 29 : Tluo e f f o c t o f v a i ' y i i ’g c o n ^ ' o n t r a t i on a o f v j ru n :

auf! (■ omul onion 1. on t r - a n . ' - f o r m a t i o n d n t i i h i t . i o n

u r - i i i g oi ir- h i r ’:'; t i t r c ' o r rum (6889 '^ weef: 2 4 )

, 178.

cells then obviously the serum cannot exert an effect* In contrast, varying complement concentrations from 1 to 10?^ resulted ir marked variations in serum TI effect. At 1% there was no TI while at 1Og TI was complete

Figur-e ■'I i 11 ustrates the morphological changes when a non-transforming f i br opapi lloma virus isolate i.s inoculated into foetal bovine cultures. This effect was identical when skin, conjunctiva, f:’olate and meningeal cultures were used„When high concentrations of a transforming virus isolate are used in conjunction with high titre antiserum, identical morphological change.s are seen. The morphological changes can be reproduced only with, high virus concentrations with antibody. The changes appear between days 5~7 post inoculation rmd are either masked or disappear by days 8-10.

Tal, F p shows the effect of prior adsorbtion of one higii titre serum (F8888 W24) using various BPV~relatei suspensions. Significantly reduced titres (P < 0.01) resulted following nd.' orbtion with CcFl purified fibropapilloma virus and tissue pieces or cells of cutaneous fibropapilloma; fibroma; BPV induced men i ngioma ; and fit-roj upi 11 ova derived- BPV-transformed fo--1al boviru' conjunctiva cells.. Slightly reduced titres (not significant) were observed following adsorbtion with normal foetal tissue; anogc-uital fibropapilloma ^0% w/v clarified susjien.sion; and teat papilloma tissue jhecco. All other adsorbants did not reduce the' TI titre of the serum.

Figure "0 illustrates a typical transformation assay plate for comparison with Figure ??. Repeated attempts todetect interference between non-transforming and transforming virus is.n1-!t.('s failed. Repeated attempts to inhibit the pheiiotyîpic appcaranc(' of trans I armed cultures using high titre TI serum wcre u n r u c c ssful even though adsorbtion with transformed ce ] 1 r, ' ! i d sip;n i icantl y reduce antibody titres.At very high cone ont rat i otrs of .anti serum ( eg neat and 1:10 dilution.'-,! tlru'e wa.s a marked inhibition of cell growth with cells becorfiinp; <-n larged, VtXCUQ bated with an almost cuboidal appearance, ho w e v e r , in those wells where cells continued to grow, they retained their transformed morphology.

, 1 7 9 ,

F ig u r e iO T r a n s fo r m a t io n assay p l a t e f o r b o v in e p a p i l lo m a

v i r u s , a) phot.ograpli o f w ho le p l a t e - row C

c o n t r o l s î d a r k e r w e l l s show t r a n s f o r m a t i o n w isps .

, 180 ,

a

Figure d1 Morphological changes seen in vitro when high concentrations of virus and high titre TI serum are used, a) affected b) control wells. These changes arc identical to tlioso produced by non-transforming fibropapilloma isolates.

. 1 8 1 .

8 «4 D is c u s s io n

A n t i v i r i o n ( v ) a n t i b o d ie s have been r e p o r t e d f o r

b o v in e p a p i l lo m a v i r u s and th e s e have been used t o v i s u a l i s e

v i r u s w i t h i n th e n u c l e i o f s u p e r f i c i a l e p i t h e H a l c e l l s i n

f i b r o p a p i l l o m a s ( S m i th ie s and O ls o n , 1 9 8 4 ) . The same a u th o r s

r e p o r t t h a t V - a n t ig e n has n e v e r been d e te c te d i n th e f ib ro m a

c e l l s and n e i t h e r has v i r u s been seen i n th e s e c e l l s un d e r th e

EM. Lee and O lson (1989) r e p o r te d a g e l d i f f u s i o n p r e c i p i t i n

t e s t f o r b o v in e p a p i l lo m a v i r u s . h f i r g - i d - u i t J c a l m ethods, as

w e l l as th o s e r e p o r t e d by K o l l e r e t a l . , (1974) and A lm e id a

and G o f fe ( 1 9 8 9 ) , no p r e c i p i t i n l i n e s deve loped when th e s e ra

used i n t h i s c h a p te r were t e s t e d . Agar and aga rose

c o n c e n t r a t i o n s were v a r ie d from O.b t o 2 ^ , b u f f e r s o f phospha te

and c i t r a t e were u se d . A n t ig e n p r e p a r a t io n s in c lu d e d

homogenised f i b r o p a p i l l o m a t i s s u e , c l a r i f i e d 20^ and 10% w /v

s u s p e n s io n s and CsCl p u r i f i e d and c o n c e n t ra te d BPV, A n t ig e n

p r e p a r a t io n s were v a r i o u s l y t r e a t e d w i t h h y p o to n ic s a l i n e ,

sodium dodec y l s u lp h a te , AO second, 2 and 10 m in u te

u l t r a s o n i c a t i o n w i t h o u t a v a i l * The c o n c lu s io n i s t h a t no

a n t i ' -V a n t i b o d ie s , d e te c ta b le by im m u n o d i f fu s io n were p r e s e n t

in. th e c a l f s e ra t e s te d *

Loo and O lson (1969b) r e p o r te d t h a t , i n e x p e r im e n ta l

c a l v e s j a n t i - V a n t i b o d ie s f i r s t appeared between 1 and 2 weeks

f o l l o w i n g i n o c u l a t i o n and p e r s i s t e d f o r up t o 16 weeks. A n t i - V

a n t ib o d y p resence i n se ra c o u ld n o t be c o r r e l a t e d w i t h th e

g ro w th o r r e g r e s s io n o f f i b r o p a p i l l o m a s .

B a r t h o ld and O lson (1974b) d e te c te d a membrane

a n t ig e n t o BPV-induced f ib r o m a c e l l s u s in g i n d i r e c t immuno

f lu o r e s c e n c e on l i v e u n f i x e d f ib ro m a c e l l s i n v i t r o . They

r e p o r te d t h a t a d s o r b t io n w i t h tum our c e l l s removed th e a n t ib o d y

a c t i v i t y i n t e s t s e r a . The c u l t u r e d f ib r o m a c e l l s were

n e g a t i v e f o r V - a r t i r e n and EM e x a m in a t io n f a i l e d t o d e te c t

BPV p a r t i c l e s . S ê i. | i /o n t ia l a n t ib o d y t i t r e s c o r r e l a t e d w i t h

f i b r o p a p i l l o m a o n s e t b u t n o t w i t h p r o g r e s s io n o r r e g r e s s io n o f

tum our g ro w th * Maximum a n t ib o d y t i t r e o b ta in e d u s in g th e

i n d i r e c t im m u n o f lu o re s ce n c e t e s t was 5 9 . Of seven c a lv e s

sam p led , 5 deve loped p e r s i s t e n t f i b r o p a p i l l o m a s , 5 deve loped

.132

s m a ll tum ours w h ich r a p id ly reg ressed and 1 showed no response

to in o c u la t io n . In two c a lv e s where fib ro m a developm ent was

m in im al and re g re s s io n was com plete 8 weeks p os t in o c u la t io n ,

a n tib o d y p e rs is te d fo r a f u r th e r 9 months a t peak t i t r e v a lu e s ,

B a r th o ld and O lson ( 1974b) d id no t a tte m p t to c o r re la te the

h is t o lo g ic a l onse t o f re g re s s io n ( t y p i f ie d by lym phocyte

a ccu m u la tio n - C hapte r 5 ) w ith a n tib o d y t i t r e s .

F o r c a lv e s 6 8 8 8 8 , 68891 and 6 8 8 8 6 i t would appear

super f i c i a l l y th a t a n tib o d y response c o r re la te s w ith the

f i r s t appearance o f f ib ro p a p il lo m a s ; however, c lo s e r in s p e c t io n

o f these c a lv e s to g e th e r w ith c a l f 68893 (F ig u re 28) shows a

p o s i t iv e c o r r e la t io n between f i r s t appearance o f lym phocyte

a ccu m u la tio n in tum ours and d e te c ta b le T I a n t ib o d ie s in the

serum* O nly c a l f 6 8 8 9 1 showed a low t i t r e w ith th e

appearance o f lym phocytes in the tum our b io p s y two weeks l a t e r .

T h is may have been a sam p ling e r r o r .

The r e s u l t s re p o r te d here in d ic a te th a t T I a n tib o d y

t i t r e s p e r s is t o n ly 4-8 weeks fo l lo w in g com ple te re g re s s io n .

These f in d in g s c o n f l i c t w ith those o f B a r th o ld and O lson (1974b)

bu t a re in g e n e ra l agreement w ith those f o r TSTA in SV40

induced tum ours (Chang e t a l , , 1977) and the f in d in g s w ith

human p a p illo m a re la te d c e l l m ediated imm'un-'ty re p o r te d by Lee

and E is in g e r (1 9 7 7 )- The h a l f - l i f e o f im m unog lobu lins in

more than 1 0 0 day o ld c a lv e s has been re p o r te d to be 2 2 , 8 days

(Cunningham, 19?8) and t h i s e s tim a te i s in accordance w ith th e

f in d in g s in t h i s c h a p te r . B la ck e t a l , , ( 1 9 6 5 ) have re p o r te d

t ra n s fo rm a t io n in h ib i t i o n t i t r e s as h ig h as 9 . 1 2 x 1 0 ' b u t malce

no comment about the complement dependence o f t h e i r te s t system ;

however th e y used non -hea t in a c t iv a te d fo e ta l b ov ine serum

(FBS) in t h e i r g row th medium. E xpe rience has shown th a t t h i s

s o u rc '" , i f fre .s ’ i can a lone supp ly enough complement fo r

t j-a n s fm ti,a tio n i n h ib i t i o n . S ince complement le v e ls in FBS a re

h ig h ly v a r ia b le , the work re p o r te d here has been conducted

u s in g 'I s tan d a rd son'"ce o f complement i.o ensure c o m p a ra b il i ty o f

t i t l e s o b ta in e d in secouenti&l assays.

. 1 8 3 ,

Adsorbtion exjieiiments reported here are in agreement v/ihh thos(-> of Barthold and Olson (1974b). They provide further evidence that while T I antibody is not directed against purified or clfi.rified BPV preparations from a variety of sources (Table 3?-) Î it is directed against in vivo and in vitro cells transf03'med by fi brnr apilloma virus. The absence of a similar reopofiec to lis: ue suspensions of teat rice grain lesions and teat papillomas is in accord with the non-cross reactivity between the TSTA’s of JCV, BKV and SV40 induced tumour cells (Padgett et al,, 1977; Padgett and Walker, 1976), Related but different papova viruses appear to have distinct TSTA's. The reduction of T I titre b y adsoj'bl ion with purified but not clarified cutaneous fibropapilloma virus may simply be a reflection of the vastly greater virus concentration in theformer than the latter. On a gram equivalent weight of tumour

3tissue, the purified extract contained 10 times greater virus than the clarified suspension * This would over-ride any equivalence between antigen and antibody as indicated in Figure 29.

Although meningoma tissue suspensions reduced TI titre in vitro, this obs*" ration does not agree with the in vivo data. At the time of post mortem (39 weeks) both calves 82 and 68891 showed large meninjfjmas but sera taken at the same time contained no detcctible TI activity. Histological examination of tumours showed no evidence of a rejection response, even though both calves had develop' d and rejected cutaneous fibropapillomas five months prc-viously. Whether this is due to an effective blood- brain barrier preventing tumour celi escape to the peripheral circulation must await further investigation.

The atseiice of TI activity in sera of calves inoculated wi th transformed cells may be due to the relative inseui. itivity of the test system since these calves were shown to be more resistant to challenge than control cell inoculated calves in vivo (Chapter 5)*-

The presence of TI antiserum and complement does not cause previously transformed 11s to revert to normal growth morphology. At high con;cut'utions, there is an apparent

. 1 8 4 .

c y t o t o x i c e f f e c t , and a d s o r b t io n w i t h t r a n s fo r m e d c e l l s does

reduce T I t i t r e . The absence o f a r e v e r s io n t o no rm a l

m o rpho log y may be due t o a n t ig e n excess i n l e s s th a n c y t o t o x i c

an t ibod y --co m p lem en t c o n c e n t r a i i ons .

8 .5 SUMMARY

T h is c h a p te r has p re s e n te d e v id e n ce f o r a complement-

dependent t r a n s f o r m a t i o n i n h i b i t i o n assay w h ich d e te c t s

a n t i b o d ie s d i r e c t e d a g a in s t B P V -tra n s fo rm e d c e l l s i n v i t r o and

BPV in d u c e d tum ou rs i n v i v o . F i r s t appearance o f a n t ib o d y

o c c u rs f o l l o w i n g f i b r o p a p i l l o m e o n s e l and i s c o r r e l a t e d w i t h

th e h i s t o l o g i c a l a j ipearance o f ly m p h o c y te a c c u m u la t io n w i t h i n

tu m o u rs . F o l l o w in g com ple te tumotrr r e g r e s s io n , T I a n t i b o d ie s

re m a in d e te c t a b le f o r 4-8 w eeks. No T I a n t i b o d ie s c o u ld be

d e te c te d i n c a lv e s i n f e c t e d w i t h t e a t r i c e g r a i n l e s i o n BPV,

t r a n s fo rm e d c e l l s o r c o n t r o l c e l l s , T I a n t ib o d y t i t r e c o u ld

be reduced by a d s o r b t io n w i t h BPV-induced meningioma t i s s u e i n

v i t r o b u t T I a n t i b o d ie s were n o t c o r r e l a t e d w i t h th e p resence

o f m ening iom as i n v i v o ,

A d s o r b t io n o f h ig h t i t r e T I a n t i s e r a w i t h t e a t r i c e

g r a i n l e s i o n t i s s u e and t e a t p a p i l l o m a t i s s u e d id n o t reduce

T I a c t i v i t y , w h i le a d s o r b t io n w i t h BPV in d u c e d f i b r o p a p i l l o m a ,

f ib r o m a and mening ioma t i s s u e and BPV t r a n s fo r m e d c e l l s caused

s i g n i f i c a n t t i t r e r e d u c t i o n s . The T I assay i s more s e n s i t i v e

th a n b u t appears, to be d e t e c t i n g s i m i l a r a n t i - c e l l a n t ib o d ie s

t o th e i n d i r e c t im m u n o f lu o re s c e n c e assay r e p o r t e d by B a r t h o ld

and O lso n ( 1 9 7 4 b ) ,

, 1 8 5 ,

CHAPTER 9 ATTEMPTS TO GROW BOVINE PAPILLOMA VIRUS IN VITRO

9.1 I n t r o d uc t i o n

Many r e p o r t s appea r i n th e l i t e r a t u r e c l a im in g t o have

grown p a p i l lo m a v i r u s e s i n v i t r o (M endelson and K l ig m a n , 1961;

M acpherson , 1962; Morgan and B a ld u z z i , 1954; Qro z la n and R ic h ,

196ip.; Noyes, 1965; E i s i n g e r e t a l » , 1975; and Hadden, 1 9 7 5 ) ,

O lson e t a l , ( i9 6 0 ) c la im e d to have grown BPV i n th e c h o r i o a l

l a n t o i c membrane o f f e r t i l e c h ic k e n eggs (see a ls o Bagdonas and

O ls o n , 1954)» A l l o f th e s e r e p o r t s a w a i t c o n f i r m a t io n » In

C h a p te r 1 ,4 » 8 , t h i s a u th o r co n c lu d e d t h a t HPV and BPV have in d e e d

been c u l t i v a t e d i n v i t r o , b u t i n e f f i c i e n t l y and un d e r e x c e p t io n a l

c i r c u m s ta n c e s . T here i s s t i l l , how ever, no r e l i a b l e method o f

c u l t i v a t i n g s e v e r a l o f th e polyoma v i r u s e s as w e l l as any

p a p i l l o m a v i r u s i n v i t r o .

T h is c h a p te r r e p o r t s on a t te m p ts made to grow BPV i n v i t r o

on a l l p r im a ry f o e t a l b o v in e c e l l c u l t u r e s e s t a b l i s h e d i n th e

l a b o r a t o r y »

9» 2 M a t e r i a l s and Methods

9 .2 .1 C e l l C u l t u r e s

S in ce a l l p a p i l lo m a v i r u s e s appear i n t r a n u c l e a r l y o n ly i n

th e n u t r i e n t s ta r v e d upper l a y e r s o f th e e p i t h e l i u m , a s ta n d a rd

method was d e v e lo p e d to t e s t c u l t u r e s f o r i n v i t r o BPV r e p l i c a t i o n .

S ince t r a n s f o r m a t i o n - s e n s i t i v i t y i s n o t a lw a ys r e l a t e d to

p e r m i s s i v i t y f o r r e p l i c a t i o n , a l l 44 c u l t u r e s e s t a b l i s h e d and

r e p o r t e d i n C h a p te r 7 were t e s te d u s in g t h i s m ethod .

Each c u l t u r e was suspended u s in g t r y p s i n / v e r s i n e and 5 x 10^3

c e l l s p lu s th e e q u i v a le n t o f 10 t r a n s fo r m in g u n i t s o f f i b r o

p a p i l lo m a d e r iv e d BPV were i n o c u la t e d i n 100 ml® BME w i t h - 10%

added FBS» B u r le r s were r o t a t e d 3 -4 t im e s p e r h o u r i n a h o t room

h e ld a t 37°C , A t w e e k ly i n t e r v a l s , th e medium was re p la c e d w i t h

100 m l . f r e s h BME w i t h 10% FBS» f o r a minimum o f 8 weekso C e l l s

were n o t s u b d iv id e d o r o th e r w is e t r e a t e d d u r in g th e e i g h t weeks®

g»2.2 E le c t r o n M ic ro s c o p y

C o l le c te d sp e n t media were p r e c i p i t a t e d w i t h e q u a l volume

added s a t u r a te d ammonium s u lp h a te a t 4^C. The p r e c i p i t a t e was

suspended i n 10 ml PBS b e fo re b e in g s u b je c te d to d i f f e r e n t i a l

c e n t r i f u g a t i o n and C^CI p u r i f i c a t i o n as d e s c r ib e d e a r l i e r f o r

v i r u s p r e p a r a t io n (C h a p te r 7 ) ® CsCI f r a c t i o n s a round 1 ,3 6 were

c o l l e c t e d , d i l u t e d to te n ml w i t h PBS and th e n c e n t r i f u g e d f o r

90 m in u te s a t 37 ,000 r . p . m , on an SW41 r o t o r i n a Beckman

,186,

u l t r a c e n t r i f u g e . P e l l e t s were resuspended i n 0 .1 ml PBS p r i o r to

n e g a t iv e s t a i n i n g f o r EM d e t e c t i o n o f v i r u s p a r t i c l e s . In a l l ,

352 s p e n t medium samples were t e s te d i n t h i s fa s h io n »

Four t r a n s f o r m a t i o n s e n s i t i v e c u l t u r e s were p re p a re d as

d e s c r ib e d unde r 9 .2 .1 b u t w i t h p a i r e d c o n t r o l b u r l e r s , and tw e n ty

5 ml p l a s t i c b o t t l e s p a i r e d w i t h c o n t r o l and t ra n s fo rm e d c u l t u r e

c o n t a in in g b u r le r s o C e l l s were removed from th e s e b o t t l e s u s in g

g la s s beads a t s e le c te d t im e s p o s t i n o c u l a t i o n and f u r t h e r

p ro c e ss e d f o r t h i n s e c t io n EM e x a m in a t io n . The t im e i n t e r v a l s

s e le c te d were 0, 0 .2 5 , 0 ,5 , 1, 2, 4 , 8, 16, 32 h o u rs ; 2, 4 , 6, 8 ,

10, 13, 17, 20, 23, 24, 28 days p o s t i n o c u l a t i o n . One o f th e s e

c u l t u r e s (5M) showed p re se n ce o f v i r u s i n th e medium 28 days p o s t

i n o c u l a t i o n , and a l l t h i n s e c t i o n c e l l samples o f t h i s c u l t u r e

were examined i n d e t a i l unde r th e EM.

One c u l t u r e (11S) was examined i d e n t i c a l l y t o t h a t o u t l i n e d

e a r l i e r . Two b u r l e r s wore s e t up as d e s c r ib e d i n 9 ,2 .1 w i t h th e

e x c e p t io n t h a t t o each medium was added 0 .5 m i l l i c u r i e s o f

t r i t i a t e d t h y m id in e . S c i n t i l l a t i o n c o u n ts were made o f a l l

CsCI f r a c t i o n s o f th e e i g h t g r a d ie n t s c o n t a in in g s p e n t medium

e x t r a c t s . G r a d ie n t samples a round r e f r a c t i v e in d e x 1 .3 6 were

examined unde r th e EM f o r BPV d e t e c t i o n . I n t h i s e x p e r im e n t c e l l s

and d e b r i s c o l l e c t e d i n th e s p e n t medium were s u b je c te d t o t h r e e

f r e e z e - th a w c y c le s and examined under th e EM (see s e c t i o n 2 . 2 , 7 ) .

P r i o r e x p e r im e n ta l t r e a tm e n t o f th e same c e l l s had shown a

c o n c e n t r a t io n o f 2 mg/ml B leom yc in t o be c y t o s t a t i c b u t n o t

c y t o t o x i c , , 2 mg/ml o f B le o m yc in was added to th e medium o f one

b u r l e r 21 days p o s t i n o c u l a t i o n .

9 . 2 . 3 T r a n s fo r m a t io n Assay

In th e th r e e cases where BPV was found i n th e medium, th e

sam ples were assayed as d e s c r ib e d i n C h ap te r 7 t o d e te rm in e th e Log

TD5D.

9 . 2 . 4 O th e r I n v e s t i g a t i o n s

The s p e n t medium fro m a l l t r a n s fo rm e d and c o n t r o l c u l t u r e s

m a in ta in e d i n th e l a b o r a t o r y were c o l l e c t e d and s to r e d a c c o rd in g

to c u l t u r e o f o r i g i n . Medium from o v e r 2 ,000 s u b d i v i s i o n s

( c o m p r is in g 40 l i t r e s ) conduc te d r o u t i n e l y was examined i n t h i s

way. Poo led s p e n t medium was t r e a t e d as d e s c r ib e d i n 9 . 2 .2 b e fo r e ,

b e in g examined f o r th e p resence o f BPV under n e g a t i v e s t a i n i n g i n th e

EM.

. 1 8 7

9 o 3 R e s u l ts

EM e x a m in a t io n o f a l l sp e n t medium fro m r o u t i n e l y s u b d iv id e d

t ra n s fo rm e d and c o n t r o l c u l t u r e s f a i l e d t o d e m o n s tra te BPV

p a r t i c l e s „

One c u l t u r e , 2M, o f t h i r t y n in e te s te d s p e c i f i c a l l y f o r BPV

g ro w th showed th e p resence o f BPV p a r t i c l e s u n d e r th e EM i n

medium c o l l e c t e d 24 days p o s t i n o c u la t i o n » F ig u r e 32 i s an

e le c t r o n m ic r o g r a p h o f t h i s i s o l a t e , O f i n t e r e s t i s th e f a c t t h a t

t h i s c u l t u r e , u n l i k e th e o th e r s , was n e g le c te d . S ince i t was

e s t a b l i s h e d o u t o f s y n ch ro n y w i t h o th e r c u l t u r e s , i t r e c e iv e d

i t i s f i r s t medium a t 3 da ys , th e second a t 10 days and th e

t h i r d a t 14 d a y s . I t was t h i s l a t t e r medium c o l l e c t e d 24 days p o s t

i n o c u l a t i o n w h ich was fo u n d to c o n ta in th e BPV i l l u s t r a t e d i n

F ig u re 32 . The c u l t u r e had s p e n t 10 days w i t h o u t medium i n

c o n t r a s t t o th e no rm a l seven f o r th e o t h e r s .

One c u l t u r e (5M) o f f o u r sampled s e r i a l l y and te s te d f o r BPV

g ro w th showed th e p resence o f BPV i n th e medium c o l l e c t e d 28 days

p o s t i n o c u l a t i o n . T h is c u l t u r e was fo u n d to be c o n ta m in a te d w i t h

S ta p h lo c o c c a l b a c t e r i a a t th e same t im e . D e t a i l e d EM e x a m in a t io n

o f s e r i a l l y sampled t h i n s e c t io n s showed BPV p re se n ce a round th e

c e l l s ; i n t r a - c y t o p l a s m i c BPV^both e n c lo s e d i n v e s i c l e s and

u n e n c lo s e d ; and between th e two la m in a e o f th e n u c le a r e n v e lo p e .

V i r u s p re se n ce c o u ld o n ly be d e te c te d up t i l l 4 days p o s t

i n o c u l a t i o n . I n a l l o f th e subsequ en t sam p les , no BPV c o u ld be

d e te c te d on t h i n s e c t io n e x a m in a t io n . No i n t r a n u c l e a r v i r u s

p a r t i c l e s were seen a l th o u g h a s m a l l p r o p o r t i o n o f th e c e l l s

showed g ro s s d e g e n e r a t iv e changes w i t h a s s o c ia te d n u c le a r

a b n o r m a l i t i e s .

In one c u l t u r e (1 1 S ) , BPV p a r t i c l e s were seen i n n e g a t iv e

s t a i n p r e p a r a t io n s o f CsCI p u r i f i e d sp e n t medium c o l l e c t e d 28

days p o s t i n o c u l a t i o n f ro m th e B leom yc in t r e a t e d b u r l e r b u t n o t

f ro m th e u n t r e a te d b u r l e r . F re e z e - th a w sam ples o f c e l l s and c e l l

d e b r i s a ls o showed th e p resence o f BPV p a r t i c l e s under th e EM,

F ig u re 33 shows th e r a d i o a c t i v i t y e xp ressed i n c o u n ts p e r m in u te

o f CsC1 g r a d ie n t s o f b o th t r e a t e d and u n t r e a te d s p e n t medium

sam p les .

T r a n s fo r m a t io n assay o f a l l t h r e e BPV sam ples fo u n d i n t h i s '

work showed a Log^^ TD5B o f 4 ,1 i n th e 2M p r o d u c t io n , compared

w i t h 4 ,3 o f th e o r i g i n a l i n o c u l a t i o n ; a t i t r e o f 2 ,7 i n th e BM

p r o d u c t io n , compared w i t h a t i t r e o f 4 ,2 i n th e o r i g i n a l in o c u lu m .

. 186 .

F ig u r e 32 E le c t r o n m ic r o g r a p h o f BPV p r e s e n t i n c u l t u r e medium 24 days

p o s t i n o c u l a t i o n m a g n i f i c a t i o n a p p r o x im a te ly x 1 8 0 ,0 0 0 .

1 8 9

o

h

§ou

1 0

8

6

4

S

2

1, 350 1, 355 1, 360 i, 365 1,370 1,375

R e fra c tive index

F ig u re 33 R a d io a c tiv ity in CS C l g rad ien ts of m ed ium

sam ples fro m B le o m yc in -tre a te d and untrea ted BPV

in fected c e ll c u ltu re s

tre a te d

u n t r e a t e d

E .M , de te rm ined BPV p o s itiv e a rea in trea ted g ra d ie n t

note: each poiiU rep re sen ts tiie mean of fo u r read ings.

. 1 9 0 .

9o4 D i s c u s s i o n

A l th o u g h many r e p o r t s i n th e l i t e r a t u r e c la im t o have grown

human p a p i l lo m a v i r u s o r b o v in e p a p i l lo m a v i r u s (see i n t r o d u c t i o n )

th e r e e x i s t s no r e l i a b l e te c h n iq u e f o r th e i n v i t r o g ro w th o f any

p a p i l lo m a v i r u s and s e v e r a l p o ly o m a v i ruses® The e v id e n c e i n

th e l i t e r a t u r e i s s u f f i c i e n t l y c o n v in c in g to c o n c lu d e t h a t

human p a p i l lo m a v i r u s has been c u l t i v a t e d i n v i t r o - b u t v e ry

r a r e l y and o n ly unde r s p e c ia l i s e d c o n d i t io n s ® ( f o r example

E i s i n g e r e t a ] . , , 1975)

The r e s u l t s o f t h i s c h a p te r t e l l a s i m i l a r s to ry® Underno

norm a l c u l t u r a l c o n d i t i o n s , ^ c u l t u r e showed BPl/ p r o d u c t io n . However

unde r c o n d i t i o n s where c e l l s rem ained i n s i t u f o r up t o two m onths ,

th r e e o f 44 c u l t u r e s t e s te d showed th e p resence o f BPV i n medium

c o l l e c t e d a t 24 o r 28 days p o s t - i n o c u la t i o n ® T h a t th e v i r u s

p re s e n t i n th e medium was BPV i s s u p p o r te d by th e EM s iz e and

m o rp h o lo g y ; th e CsCI d e n s i t y ; and th e i n v i t r o t r a n s f o r m a t io n

by two o f th e t h r e e i s o l a t e s . S u p p o r t f o r t h e f a c t t h a t th e v i r u s

was p roduced by th e c a l l s comes from th e r a d i o a c t i v i t y peak a t th e

BPV r e f r a c t i v e in d e x o f 1«36 i n d i c a t i n g t h a t th e v i r a l DIMA had

in c o r p o r a t e d t r i t i a t e d th y m id in e w h ich can o n ly o c c u r d u r in g

v i r a l p r o d u c t i o n . The i n a b i l i t y to d e te c t BPV i n t h i n s e c t io n

samples may be a r e f l e c t i o n o f th e v e ry s m a l l number o f c e l l s

w h ich were a c t i v e l y p e rm is s iv e f o r DMA r e p l i c a t i o n . C om pa ra t ive

t r a n s f o r m a t i o n t i t r e s o f i n o c u la t e d v i r u s and p roduced v i r u s

i n d i c a t e t h a t th e v i r u s p roduced was i n v e ry lo w q u a n t i t i e s .

F u r t h e r e v id e n c e t h a t th e v i r u s was a c t u a l l y p roduced i n v i t r o

comes f ro m th e d i l u t i o n f a c t o r s i n v o l v e d . By th e t im e v i r u s was

fo u n d i n s p e n t medium, th e o r i g i n a l in o c u lu m had been d i l u t e d by

s u c c e s s iv e medium changes by a f a c t o r o f 10^ , I t was c o n s id e re d

p o s s ib le t h a t th e v i r u s c o u ld be absorbed b u t n o t degraded by

th e c e l l s and th e n r e le a s e d on t h e i r d e a th . The r a d i o l a b e l l i n g

e x p e r im e n t m i t i g a t e s a g a in s t t h i s , how ever.

The w ork in v o lv e d i n c a r r y i n g o u t th e e x p e r im e n ts was

c o n s id e r a b le 6 The r e s u l t s a re a t b e s t a s m a l l c o n t r i b u t i o n to

p o s s ib le avenues f o r f u r t h e r w o rk . They a re by no means c o n c lu s i v e ,

and w i l l n o t bo p u b l is h e d e x c e p t i n t h i s t h e s i s . The two f a c t o r s

w h ich a l l t h r e e p ro d u c tv e " b u r s t s " have i n common a re ( l )

p ro lo n g e d c o n t a c t i n h i b i t i o n and (2 ) s e ve re s t r e s s e i t h e r th ro u g h

la c k o f n u t r i e n t s , b a c t e r i a l c o n ta m in a t io n o r B le o m yc in t r e a t m e n t .

. 1 9 2 ,

9 .5 Summary

Three o f f o r t y - f o u r c u l t u r e s t e s te d showed e v id e n c e o f i n

v i t r o v i r a l r e p l i c a t i o n between 24 - 28 days f o l l o w i n g BPV

i n f e c t i o n . These c u l t u r e s were s u b je c te d to p ro lo n g e d c o n ta c t

i n h i b i t i o n and se v e re s t r e s s p r i o r t o th e appearance o f BPV

i n th e medium. D e t a i l e d t h i n s e c t io n EM e x a m in a t io n f a i l e d to

d e t e c t v i r a l p a r t i c l e s i n th e c e l l s f ro m one c u l t u r e show ing

BPV i n th e medium and f r e e z e - th a w samples o f c e l l u l a r d e b r i s * De

s p i t e th e amount o f work i n v o l v e d , th e r e s u l t s a re i n c o n c lu s i v e

and may o n ly s e rv e as s u g g e s t iv e avenues f o r f u r t h e r re s e a rc h

e f f o r t .

. 1 9 3 ,

CHAPTER 10 GENERAL DISCUSSION AMD MAIM THESIS FINDINGS

No p r e v io u s s u rv e y s o f t e a t o r cu taneous B P V - re la te d l e s io n s

have been p u b l i s h e d , A s u rv e y o f a l im e n ta r y p a p i l l o m a t o s i s has r e p o r te d

an o v e r a l l p r e v a le n c e o f 19% ( J a r r e t t e t a l . , 1 9 7 8 ) , In the human,

p re v a le n c e f i g u r e s f o r p a p i l l o m a t o s i s , f o c a l e p i t h e l i a l h y p e r p la s ia

and o t h e r H P V - re la te d l e s io n s v a ry w id e ly a c c o rd in g t o se x , age and

g e o g ra p h ic a l p a ra m e te rs ( S e c t io n 1 . 3 ) , The t e a t s u rv e y r e p o r te d i n

C h a p te r 2 p r o v id e s an o v e r a l l p re v a le n c e o f 369^^ i n 721 c a t t l e

i n s p e c t e d . T here were s i g n i f i c a n t sex s i f f e r e n c e s i n th e p re v a le n c e

o f th e d is e a s e and n u U ip a r o u s fe m a le c a t t l e had s i g n i f i c a n t l y fe w e r

t e a t s a f f e c t e d th a n pa rous fe m a le c a t t l e . These f i n d i n g s c o n t r a s t

w i t h th o s e o f G a r r e t t e t a l . (1978) who r e p o r te d no s i g n i f i c a n t sex

d i f f e r e n c e s i n th e p re v a le n c e o f a l im e n t a r y l e s i o n s . The h ig h

p re v a le n c e o f t e a t l e s i o n s was s u r p r i s i n g because c o n c u r r e n t exam in

a t i o n o f th e r e s t o f th e in te g u m e n t showed a cu ta n e o u s f i b r o p a p i l l o m a

p r e v a le n c e o f 2.71% , These d i f f e r e n c e s may have been due to a n im a ljt'atl b«,

f a c t o r s (e g , th e t e a t . an im m u n o lo g ic a l ly d e p r iv e d s i t e , o r , th eA

t e a t i s more r e g u l a r l y t r a u m a t is e d th a n o th e r p a r t s o f th e in te g u m e n t )

o r v i r a l d i f f e r e n c e s ^ H i s t o l o g i c a l e x a m in a t io n o f i n d i v i d u a l l e s io n s

showed t h a t t e a t l e s io n s c o u ld be d iv id e d i n t o t h r e e b road c a te g o r ie s -

f r o n d e d t r u e p a p i l lo m a , f o c a l e p i t h e l i a l h y p e r p l a s t i c l e s i o n s ( r i c e

g r a i n ) and f i b r o p a p i l l o m a (C h a p te r 3 ) . The e p id e m io lo g ic a l da ta

p re s e n te d i n C h a p te r 2 i n d i c a t t h a t a t l e a s t two v i r u s / h o s t

i n t e r a c t i o n s o c c u r r e d . The t e a t r i c e g r a in l e s i o n s were s i g n i f i c a n t l y

d i f f e r e n t i n d i s t r i b u t i o n b o th i n shape o f c u rve and number o f l e s i o n s .

D i f f e r e n c e s were a ls o d e te c te d i n v i r u s p a r t i c l e d ia m e te r and a p p a re n t

m o le c u la r w e ig h t o f m a jo r v i r a l c a p s id p r o t e i n .

When s i n g l e ca s e , s im p le l e s io n t y p e GPU e x t r a c t s were t r a n s m i t t e d

to e x p e r im e n ta l c a l v e s ,m o r p h o lo g ic a l and d e v e lo p m e n ta l d i f f e r e n c e s

between th e t r a n s m i t t e d le s io n s were c o r r e l a t e d w i t h th e l e s i o n ty p e o f

th e o r i g i n a l e x t r a c t s , BPV was e x t r a c te d from a l l t r a n s m i t t e d l e s i o n

t y p e s . K o c h 's p o s t u la t e s were th u s f u l f i l l e d - and f o u r s e p a ra b le BPV

e x t r a c t s were i d e n t i f i e d , - c u ta n e o u s , a n o g e n i t a l f i b r o p a p i l l o m a ;

t r u e p a p i l lo m a and r i c e g r a in l e s i o n . C h a l le n g e e x p e r im e n ts re v e a le d

t h a t i n v i v o r e s i s t a n c e to r e i n f e c t i o n w i t h f i b r o p a p i l l o m a e x t r a c t s

was n o t accom panied by r e s i s t a n c e to i n f e c t i o n w i t h BPV f ro m p a p i l lo m a

o r r i c e g r a in l e s i o n s . S i m i l a r l y r e g r e s s io n o f f i b r o p a p i l l o m a on c a lv e s ,

was n o t accom panied by r e g r e s s io n o f o t h e r l e s i o n t y p e s .

I n C h a p te r 5, e v id e n c e i s p ro v id e d f o r d i f f e r e n c e s i n a b i l i t y t o

in d u c e m ening iom as o f BPV e x t r a c t s . A l th o u g h a l l v i r u s i n t r a c e r e b r a l l y

i n o c u la t e d c a lv e s showed l e s io n s a t i n t r a d e r m a l i n o c u l a t i o n s i t e s , o n ly

. 1 9 4 .

th o se c a lv e s in o c u la t e d w i t h f i b r o p a p i l l o m a d e r iv e d BPV deve lo p e d

m en ing iom as . The t e a t r i c e - g r a i n l e s i o n d e r iv e d BPV in o c u la t e d c a l f

d id n o t show mening iom a development®

Even th ough th e t o t a l number o f e x p e r im e n ta l c a lv e s used was

s m a l l , th e above m e n t ioned e x p e r im e n ts s u g g e s t two b ro a d v i r u s

c a te g o r ie s e x i s t . One ty p e w i t h a c e l l u l a r t r o p h is m w h ich in c lu d e s

b o th e p i t h e l i a l and mesenchymal t i s s u e s and th e o t h e r w h ich i s l i m i t e d

to e p i t h e l i a l t i s s u e s o n ly * W i t h in th e s e two c a t e g o r i e s , o t h e r

d i f f e r e n c e s e x i s t b o th i n th e ty p e o f e p i t h e l i a l tum ou r p roduced and

th e degree o f mesenchymal f ib r o m a in v o lv e m e n t .

A l th o u g h th e l i t e r a t u r e now c o n ta in s ample e v id e n c e f o r th e

e x is te n c e o f s e p a ra te human p a p i l lo m a v i r u s e s , some a s s o c ia te d w i t h

s p e c i f i c d is e a s e syndromes - o n ly one pape r r e p o r t s th e e x is te n c e o f

a non f i b r o p a p i l l o m a l e s i o n i n c a t t l e ( B a r t h o ld e t a l . , 1974 )*

The te rm s usod i n t h i s t h o s i s to deno te d i f f e r e n t t e a t l e s io n s

a re th o s e w id e l y used , b u t n o t r e p o r t e d i n th e l i t e r a t u r e * I t i s

p ro b a b le t h a t th e te rm s were f i r s t used by D r . Ia n Laude r (M* M urray -

p e r s o n a l c o m m u n ic a t io n ) . T h e r e fo r e , th e e x is te n c e o f d i f f e r e n t t e a t

l e s i o n ty p e s i n c a t t l e has been w id e ly r e c o g n is e d , even i f n o t d u ly

r e p o r t e d , i n t h i s d e p a r tm e n t* D i f f e r e n t i a l e x p e r im e n ta l o n c o g û n ic i t y

among i s o l a t e s o f th e same p a p o v a v i ru s has o n ly r e c e n t l y been shown

(P a d g e t t e t a l . , 1977) f o r GCV v i r u s , however th e e x i s t e n c e o f

d i f f e r e n t o r a l cu ta n e o u s p a p i l lo m a v i r u s e s i n th e same s p e c ie s has

been g e n e r a l l y a c c e p te d f o r a lo n g t im e (eg* human, s im ia n , e q u in e ,

ca n in e and r a b b i t species)» The b o v in e s p e c ie s can now be p la c e d w i t h

th e s e o th e r s i n t h a t i t possesses d i f f e r e n t p a p i l l o m a v i r u s e s .

F u r t h e r e v id e n c e f o r th e e x is te n c e o f d i f f e r e n t p a p i l l o m a v i r u s e s

comes f ro m th e i j j v i f c r o work r e p o r t e d i n C h a p te rs 7 and 8* F ib ro p a p

i l l o m a d e r iv e d BPV f ro m c u ta n e o u s , a n o g e n i t a l , a l im e n t a r y and t e a t

l e s i o n s t r a n s f o r m f o e t a l b o v in e c e l l s i n v i t r o , w h i l e BPV from

p a p i l lo m a and t e a t r i c e g r a in l e s i o n s do n o t * I n h i b i t i o n o f i n v i t r o

t r a n s f o r m a t i o n i s complement depen dan t* T r a n s fo r m a t io n i n h i b i t o r y

( T l ) a c t i v i t y c o u ld be d e te c te d i n s e ra f ro m c a t t l e w i t h f i b r o p a p i l l o m a s

b u t n o t i n c a t t l e w i t h r i c e g r a in l e s io n s *

In v i t r o t r a n s fo rm e d c e l l s were t u m q r ig e n ic i n nude m ice b u t n o t

c a t t l e . However th e y d id in c r e a s e i n v iv o b o v in e r e s i s t a n c e to c h a l le n g e

w i t h f i b r o p a p i l l o m a d e r iv e d BPV b u t n o t p a p i l lo m a o r r i c e g r a i n l e s i o n

d e r iv e d BPV*

T h is t h e s i s th u s p r o v id e s h i s t o l o g i c a l , e p id e m o lo g ic a l , im m uno log

i c a l ( i n v i v o and i n v i t r o ) , p h y s i c a l and b io c h e m ic a l e v id e n c e f o r th e

> 1 9 5 ,

e x is te n c e o f d i f f e r e n t ty p e s o f BPV. F u r t h e r work on BPV-DNA w i l l be

a b le t o d e f in e these d i f f e r e n c e s a t th e m o le c u la r l e v e l . W h i le

c o m p le te ly s p e c u l a t i v e , i t i s p o s s ib le t h a t BPV d i f f e r e n c e s r e p o r te d

he re c o u ld have re le v a n c e to th e q u e s t io n o f w h ich v i r u s ty p e ( s ) a re

in v o lv e d i n th e m a l ig n a n t B P V - re la te d le s io n s seen i n th e b o v in e

upper a l im e n t a r y t r a c t .

In v i t r o t r a n s f o r m a t i o n by BPV has been r e p o r t e d e a r l i e r ( s e c t i o n

1 * 4 .7 ) and t h i s t h e s i s e x te n d s know ledge i n d e f i n i n g th e p r e c i s i o n

and s e n s i t i v i t y o f t h i s phenomenon f o r use as an a l t e r n a t i v e method o f

q u a n t i t a t i n g f i b r o p a p i l l o m a BPV b i o l o g i c a l l y . The t r a n s f o r m a t i o n -

s e n s i t i v e t i s s u e s o f th e b o v in e fo e tu s a re a ls o r e p o r t e d as m en inges ,

c o n j u n c t i v a , p a la t e and s k i n . T r a n s fo r m a t io n t i t r e s v a r ie d w i t h v i r u s

c o n c e n t r a t i o n s and n o t f o e tu s age o r t i s s u e ty p e fro m w h ich th e c u l t u r e

was i s o l a t e d . T ra n s fo rm e d c e l l s showed h e r i t a b l e changes, an

in c r e a s e d l i f e s p a n and were t u m o r ig e n ic i n nude m ic e . They a ls o

in c re a s e d i y ( / i v o r e s i s t a n c e o f c a lv e s to subsequ en t c h a l le n g e w i t h

f i b r o p a p i l l o m a BPVo

T r a n s f o r m a t io n i n h i b i t i o n ( T l ) has been shown t o be a com p lem ent-

dependan t a n t i - t r a n s f o r m e d c e l l phenomenon i n t h i s t h e s i s . A l th o u g h

r e p o r t e d e a r l i e r ( B la c k e t a l . , 196 3 ) , t h i s t h e s i s e x te n d s know ledge

by d e f i n i n g th e p r e c i s e r e q u i re m e n ts f o r th e s u c c e s s fu l use o f T I as

an assay sys tem m easu r ing a n t i B P V - tra n s fo rm e d c e l l a n t i b o d ie s i n th e

s e ra o f c a lv e s show ing c e r t a i n B P V - re la te d tu m o u rs . The c e l l u l a r

a n t ig e n s d e te c te d u s in g th e T I assay appear to be s i m i l a r to th o se

r e p o r t e d by B a r t h o ld and O lson ( l9 7 4 b) and may be TSTA's as

d e s c r ib e d i n o t h e r p a p o v a v i ru s sys tem s ( s e c t i o n 1 . 4 . 1 ) . T I a n t ib o d ie s

were f i r s t d e te c te d i n th e s e ra o f f i b r o p a p i l l o m a b e a r in g c a lv e s

c o i n c i d e n t w i t h th e f i r s t a p p e a r a n c e o f ly m p h o c y te a c c u m u la t io n

i n t i s s u e b io p s y samplesbaing d e te c te d # T I a n t i b o d ie s p e r s i s t e d f o r

4 -8 weeks f o l l o w i n g co m p le te r e g r e s s io n o f f i b r o p a p i l l o m a s .

The d e t e c t i o n o f BPV p a r t i c l e s i n e x t r a c t s o f m i l k pu rchased from

r e t a i l o u t l e t s p rom pted an i n v e s t i g a t i o n o f t im e - te m p e r a tu r e t r e a t m e n t

on BPV i n v i v o and i n v i t r o . P a s t e u r i s a t i o n was shown i n v i v o and i n

v i t r o t o have no e f f e c t on BPV a c t i v i t y . In v i t r o , co m p le te i n a c t i v a t i o n

o f BPV t r a n s f o r m a t i o n o c c u r re d a f t e r 30 m in u te s a t 80 C and a t e n f o l d

r e d u c t i o n i n t i t r e f o l l o w e d t r e a tm e n t f o r 4 h o u rs a t 60°C , BPV i s th u s

s i m i l a r t o m ost, b u t n o t a l l p a p o v a v i ru s e s as l i s t e d i n 1 . 6 ,3 i n i t s

r e l a t i v e h e a t s t a b i l i t y . The r e s u l t s o f t h i s w o rk , c o u p le d w i t h th e

h i s t o l o g i c a l and g ro s s s i m i l a r i t y o f l e s io n s o f th e b o v in e t e a t and '

c e r t a i n o r a l l e s i o n s i n man make f u r t h e r i n v e s t i g a t i o n o f p o s s ib le

. 1 9 6 ,

human l e s i o n s r e l a t e d to BPV o f d i s t i n c t im p o r ta n c e even though

l i t e r a t u r e e v id e n c e f o r BPV i n f a c t i o n i n man i s e q u iv o c a l ( s e c t i o n 6 . 4 ) *

S u b s t a n t ia l e f f o r t made to f i n d a method f o r c u l t u r i n g BPV i n v i t r o

was l a r g e l y u n re w a rd e d . T h is a u th o r can r e p o r t t h r e e o c c a s io n s o f 44

a t te m p ts where BPV was fo u n d i n c u l t u r e medium between 24 - 28 days

p o s t i n o c u l a t i o n . I t appea rs t h a t p ro lo n g e d c o n t a c t - i n h i b i t i o n

c o u p le d w i t h se v e re s t r e s s im m e d ia te ly p r i o r t o v i r u s r e le a s e were

c o n t r i b u t o r y f a c t o r s . As w i t h o t h e r a u th o rs ( s e c t i o n 9 , 4 ) , t h i s work

i s f a r f ro m c o n c lu s iv e and, a t b e s t , may p r o v id e id e a s f o r f u r t h e r

re s e a rc h e f f o r t .

The main f i n d i n g s o f th e work r e p o r t e d i n t h i s t h e s i s a re as

f o l l o w s :

1) The deve lo p m e n t and c h a r a c t e r i s a t i o n o f an i n v i t r o t r a n s f o r m a t i o n

assay f o r f i b r o p a p i l l o m a d e r iv e d BPV.

2) The deve lo p m e n t o f a complement dependan t t r a n s f o r m a t i o n -

i n h i b i t i o n assay d e t e c t i n g a n t i ~ f i b r o p a p i l l o m a d e r iv e d BPV-

t r a n s fo rm e d c e l l a n t i b o d ie s *

3) The i s o l a t i o n and t r a n s m is s io n o f d i f f e r e n t b d v in e p a p i l lo m a v i r u s e s *

These v i r u s e s a re d i f f e r e n t i n p h y s i c a l , b io c h e m ic a l , im m u n o lo g ic a l

and b i o l o g i c a l c h a r a c t e r i s t i c s *

4) The d e f i n i t i o n o f th e h e a t - s e n s i t i v i t y o f BPV i n v i t r o and i n v i v o .

5) The a b a t t o i r s u rv e y o f t e a t l e s i o n s w i t h th e d i s c o v e r y o f h ig h

p re v a le n c e s and p a r t i c u l a r e p id e m io lo g i c a l f e a t u r e s o f d i f f e r e n t

l e s i o n t y p e s ,

6 ) The f i n d i n g t h a t f i b r o p a p i l l o m a d e r iv e d B P V - t ra n s fo rm e d c e l l s a re

tu m o L ir ig o n ic i n nude m ice b u t n o t c a t t l e . However, c a t t l e a re more

r e s i s t a n t t o su b se q u e n t c h a l le n g e w i t h f i b r o p a p i l l o m a d e r iv e d BPV

f o l l o w i n g i n o c u l a t i o n o f t r a n s fo rm e d c e l l s ,

7 ) The d is c o v e r y o f c e r t a i n f a c t o r s w h ich may be in v o lv e d i n . a f u t u r e

s e a rc h f o r a method o f i n v i t r o c u l t i v a t i o n o f BPV,

. 1 9 7 ,

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' 0 7 o

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24 1

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. 242

APPRND]) A - R E S U L T S Of

(i) W H O L E A N I M A L DATA -

A B BATTOIR

- S U M M A R Y

PAPILLOMA, S U R V E Y

SILBSL’RVEY N U M B E R C A T T L E E X A M I N E R

C A T T L EA F F E C T E ID

T E A T SA F F E C T E D

1-9-77 h ‘J H e i f e r 21 33.3 41 65.14 9 G ow 2 2 44.9 4-6 93.979 Ma, L(' 10 25.6 14 35.9

Ü-9-7Y 4 9 [leifcj- 1.-1 3 2 .6 31 72.1102 Cow 45 44.1 117 1 1 5 . 0

2 8 Ma,le 6 2 1 . 4 7 2 5 . 0

15-9-77 2 7 H e i f e r 11 40.7 28 1 0 4 , 0

68 C o w 34 50.0 77 1 1 3 . 0

38 Mal(- 6 15=8 10 2 6 . 0

29-9-77 37 H e 1 JU r 10 2 f.O 20 5 4 . 0

41 Co w 2 0 4 8 . 8 49 1 1 9 . 0

2 7 Ma,le 2, 1 4 . 8 4 1 4 . 8

6-10-77 56 H e i f e r 12 2 1 .4 21 3 7 . 5() 8 Cow 52.9 76 1 1 1 . 83 5 M,a.le 9 2 5 . 7 14 4 0 . 0

Note: only j n n l m u le hud Gcrolal papillomas ; rest wereon teats of a n i m a l s of both sexes. Scrotal p u pil l o ni as no 1 1 n c 1 u d e d in a urn ni a ry table.

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A P i ‘P l i ) [ X A

( i l l ) THAT D A T A - - APTAI i . JIJBAURTEY OP I / 9 / 7 7

: UMB E R NlJniH:!.; PROD A

H U M B E R PAir OR ROUND

DUMBER R.rc J OR AIK

T O T A L N U M B E R LESIO N S

I 1. 0 0 1

0 10 14

> Ü 1 9 10

4 A L 0 1

') 1 5b 1 0 0 ].

7 A 0 ], U0 1. 4 77 I (1 4 4

10 4 711 2 0 0 41 .' 0 0 2

1 ; 0 0 1 1

14 1 0 0 1

lU c' 0 0 2l b L 0 1 2

1 / 2 A 718 L .1. 0 2

17 I (. 0 1AO 1 0 0 1A1 .1 0 1 22 A I 0 0 1

14 1 0 1 2

14 1 0 7 8A U I. 0 4 4

' i ' i 4 6-

■•’ 7 ■1 L) 0 4A'8 0 0 9 9A 9 L 0 14 24

40 0 0

41 1 0 2 5

52

A Pi'EH D I X A

(ill ) T E A T DATA - B E T A !:., SUBSURVEY OP 1 / 9 / 7 7

N U M B E R

14

i'ji''47

48

49 4 0

4 1

4 2

4 4 44

44

4 b 4 ) ' 4 844

40

41

42

4 4 44 44 4 b

4 7 48

59 6 0

h i

bb

N 4, 0 0 : 1

PROND

u 1 J 0

( ' i 0

0

0

0

I

0

J. 0

0

0 ri

0

0

0

u 0

j.

0

0

1

0

1

1

0

MUMHER UR HU"N

NUMBER R i C G R A I N

TOTAL NUMBER LESIONS

110

1.1

14

0

1

1

0

0

0

0

1

0

2

0

0

b

0

1

1

2

441

0

6

0

0

4

10

1

51

00

0

41

0

b141

2

2

45 1

2

71545 1 2

1

2

2

52

1

2

1

42

2

253

APl'ËNDIX A.

( i l l ) TEAT DATA - DOTAP, SÜB3URVEY OF I / 9 / 7 7

N U M B T O N U M B K x U U M B E K 1 0. 0 ' N U M B E R T O T A L N U M B E RFROND OR HORN:) HI CE G R A I N L E G I O N S

0 1 0 0 1b/1 1 0 0 1fjb (■' 1 Ü 7

1 2 6b 76 8

01

00

16

17

Ob' O' 0 270 1 0 0 171

7:'0(1

:l0

2 46

( ' ) 8 0 4 1274 0 1 5 4-77 0 0 6 67b 0 1 1 277 0 0 4 4'78 0 i 2 4' ( ' ) 0 0 4 48 0 0 0 1 181 1 0 0 1BE 0 8 1 4BE 1 4 684 0 L 2 3BE 1 1 48b 0 1 4 487 0 0 288 L 1 0 2BE 'i 28 44EO 0 18 20'El 0 41 449E 0 0 12 12E i 1 il 16 19

254

A PJ-ENDI X A

( i i i ) TEAT DATA - P K r A l c SUBSURVEY OF 8 / 9 / 7 7

N i m S E R NUM B E R N U M B E R 1 0, P NU M B E R T O T A L N U M B E RF R O N D uR H O U N D R I C E G R A I N L E S I O N S

i 0 5 50 1 4

0 0 5 '4u 0 1 1 2

1 4 6h 1 0 b 47 0 J. 1 ?8 0 1 0 ].

9 L 0 1.10 0 1. 311 1 6 91 ' 1 0 0 1

1) 11 I b 414 0 0 41 ‘; 0 0 ?1 b L 0 0 1

17' 1 0 0 1]. 8, O' 0 O

1 ' » 1 1 1 4■ : 0 1 Ü 9

/ 1 J. 0 1 22,-1 0 0 4 32 4 0 0 6 6

2 4 1 0 32 7 4 0 0 3, ' ' ) 0 4- Ô

2 ( 1 0 4 4, ’ 8 1 () b 4

2 b 0 0 4 4

7 0 0 1 4 6

4L 1 L 1 44b 1 0 0 ].

255

API ’EN DIX A

; i i i ) TEAT DAT A — .l'i'jl1Ü 8 3 U B G U R V E Y OE 8/9/77

N U M B E R NJ7 8Ei N U M B E R PL/r N U M B E R T O T A L N U M B E RFROND OR ROUND H ICE G R A I N L E S I O N S

44 i ’ 0 0 2

44 '' i 4 6

47 ,] 0 447 0 2 1 447 J. 0 4 748 i I 4 747 i 4 12 16

4 0 i P' 5 8

41 1.0 0 0 10

4P 0 0 44 4 7 0 0 744 b' 1 5 11

4 ‘j 0' 17 174 8 (1 l 7 84 / 0 0 6 64 8 0 L 5 44 7 0' 0 2

70 (’ il 1 47-1 I 9 12

7P p L 4 774 1 0 0 1

74 ]. 0 2 47 7 8 0 0 27 b 0 0 2

7 7 J. ü 1 2

78 ' 0 2

77 i 0 4 5o 0 i 0 0 1bi i 0 162 0 0 7 78 4 l) ‘j 4 76 4 0 L 0 1

56

APi 'ENDIX A

TJUAT J)A r 7 0'.' 0 I, ;>UB0URVE'7 OP 8/ 9 / 7 7

rMBER N IlMHKb H U M B E R Pi.bl D U M B E R TOT.AL N U M B E R

----------------—

RuHO OR R O U N D R I C E G R A I N L E S I O N S

8 7 0 4 4i J t j 0 4 467 0 1 4 4OÜ y 0 4 46 7 [ 0 ;i 2

7 0 b 0 1 471 0 6 a72 0 i 0 1

74 O' 0 1 1

74 1. 1 4 6

7 b 0 b a78 0 5 577 ! I 4 978 0 ' 4 777 0 8 10 12

80 j 1 0 2

8 L 1 0 0 1

8 b 0 4 784 i 1 4 6

84 1 6 9

87 0 ;> 0 2

8 b 0 1 8 9

87 0 1 5 688 0 1 4 587 0 1 1 2

7 0 0 0 6 8

71 0 (4 6 6

7.-' 0 1 474 0 1 6 774 1 0 0 1

7 7 0 1 0 1

78 0 0 4 47 7 0 .1 0 1

2 57

APPENDI X A

( i i i . ) TEAT DAI\\ "_DETAi L SUBSURVEY OP 8 / 9 / 7 7

UMB E R N U M B E R N U M B E R E7\7 NUMBER TOl’AL N U M B E RP R O U D UR ROUND H I C E C R A I N L E S I O N S

78 J. 0 0 177 0 0 4 4

100 0 I 4 5lOL 0 2 0 2102 0 2 5104 0 0 2104 0 I 0 1107 0 0 2107 1 1, 0 2

107 0 1 4108 1 0 6 7107 1 1 ) 1 2110 0 7' 0 5L Li 1 L) 0 1112 1 0 0 1

114 1 0 0 111/j 0 4 7117 1 20 24117 0 i. 0 1117 1 1 4 5118 0 1 2 3117 0 0 212 0 t. I) 0 212 1 i 1 12 1412,-' 0 7 0 6

-1 ' 4 2 L 40 4412 U 0 j 0 412 7 0 0 4 3-I ' b , 0 0 8 8i: -' i 2 0 0 412 8 b I 40 5112 7 0 4 0 4

2 5 8

APPENDIX A

( i i i ) TEAT d a t a - h P r A I i . SUB3ÜRVEY OF 1 7 / 9 / 7 7

N U M B E R NIP.IBE-: N U M B E R F!. Al' N U M B E R T O T A L N U M B E RF R O N T OR ROU N D HICE G R A I N L E S I O N S

1 8 0 0 8

0 9 104 0 / 6

'1 1 0 0 1

7 0 1 2 ^

o 1 0 0 1(■’ 0 2 7 7

8 / 0 0 2

9 0 4 0 7

10 0 2 4 711 0 , ’ 7 5

1/ ) 1 10 14

1 i J- O' 0 1

IN 1 1 4 6

1' - 1 0 b 9

l b I 0 0 1

I V '! 0 7

10 0 7 7

19 4 ' 6 11

2 0 4 0 4 7

1 4 0 0 4

22 47 0 a 4404 11 1 9 21

24 7 2 a 17

27 0 1 0 1

2b 0 4 6 10

27 1 0 4

28 0 0 4 L

29 2 1 4 7

30 2 I 10 17

259

AP. ’EKÜIX A

T EAT J7Ar 7 G U B G U R V E Y OF 17/ 7 / 7 7

MB EH NU,.: il': i N U M B E R F 7.,’ NUMBER T O T A L N U M B E R

— — —

F R u N j' üR HO UNI) HicE G RAID L E S I O N S

41 I 0 0 132 - 0 4 74 3 L 0 4

44 0 6 8

3 7 / 1 0 43b L L 1 44 7 L 0 0 1

4 H (. _ ' 4 7

47 1 ) :i 0 1'1 0 L 1 4 7

41 C > 4 0 4

4 2 l : 0 1 144 0 0 2

4 4 0 V 4 6

4 7 J. 0 2 44o J. 0 2 44 7 0' J. 4 54 8 0 0 6 6

47 0 1 4 470 2 0 1 3bl L 0 1 2

72 0 0 2

')4 1 1 0 2

74 0 1 1 2

77 0 4 0 4

77 i. 0 477 I ü 0 1

78 I 0 4-

79 u 1 1 2

60 u 0 4 4u I 0 0 2 2

6;' 0 2 0 2

2 6 0

APPENDI X A

( i i i ) TKAT DAT \ - ])LP\AL_u SUBSURVEY OF 17/ 9 / 7 7

N U M B E R NUMiblRFROND

N U M B E R PI OR HOUND

a' N U M B E RRICE G R A I N

T O T A L N U M B E R L E S I O N S

6 4 L 1 0 2

64 0 0 2

bb 1 L 7 7

bt) I 0 0 1

b 7 1 0 1 2

a 8 1 0 0 1

bb 0 1 1 2

70 0 ■'1 2 6

71 0 0 2 2

Y a 1 0 0 1

74 Ü 1 9

74 L 0 0 1

77 0 0 1 1

7 b 0 1 0 1

77 1 0 1 2

78 1 0 4 7

79 L 0 0 1

80 0 4 0 4

81 0 ; 0 6

80 18 0 2 20

64 11 0 0 11

2 6 1

ADPEN D I X A

( i i i ) SÜBSURVEY OF 2 9 / 9 / 7 7

N U M B E R N U M B E R H U M B E R la.v' NUM B E R T O T A L N U M B E RF R O N D OR HOUND RIC G R A I N L E S I O N S

1 1 4 4 82 1 '1 15 204 / 0 4 6

4 0 1 1 21 5

h J. 4 4 8

( 0 1 2 4H 0 21 6 69 0 1 410 0 0 1 111 1 0 2 412 0 0 5 414 ]. 0 0 1

14 1 0 1 2

14' 1 1 1 4lu .1 0 0 11 1 0 4 5lb 2 0 0 219 ' 0 0 220 / 1 4 621 0 2 2 42 2 1 b 1 42 4 0 1 424 2 1 1 42 4' 0 0 1 1

1 2 1 2/ il 1 4

. ' b 2! 2 1 129 1. 1 0 24 0 1 4 641 L 0 0 142 2 0 0 24 4 0 •Î 1 5

262

A PPENDIX \

(iii ) SUBSURVEY OF 29/9/77N U M B E R N U S H U M B E R F R W N U M B E R T O T A L N U M B E R

F R O N D OR R O U N D HICE G R A I N L E S I O N S

O L 4 .

0 1 2 5

J

u I

8

4 7 0 1 2 4

4 8 .. 0 0 2

4 9 () 1 0 1

/jO . 10 18 27

/| 1 0 0 1 14 : [ 0 0 1

44 ( ^ 0 4 6

4 4 I 4 9 17

1 4/ ] b 1 0 0 14 7 2 , 0

/j 8 ' ' 4 64 9 0 0 2

7 0 O' 1 0 1

7 ]. 0 1 0 1

7 0 12 0 18 4 0

7 4 7 0 12 49

7 4 1 7 0 2 2 49

77 54 0 8 41

7 b 4 4 8 16

7 7 0 4 7 97 8 0 4 2 6

7 9 0 l./i 6 20

bO 0 4 b 10

61 1 4 6

f>2 () t) 8

63 0 1 4 7

u4 0 4 1 4

u ‘.J 0 8 14

' I ' I ' i o 8 1 4

4 14

264

APPENDIX A

(iii) T E AT D AT \

N U M B E R NUl.PEPF PON a UK HOl'NiJ

SUBSURVEY OF 6/IO / 7 7

N U M B E R T O T A L N U M B E RR I C E G R A I N LESIONS

1 L 1 1 31 0 0 1

> 2 0 0 54 0 0 2

I 0 6h 1 0 0 1Y 0 0 2b L 0 39 3 0 0 51 0 '' ' J 0 f)

I 1. (J J 0 -<

i,: 0 <.) 0 919 0 6 0 5la 0 1 319 0 2 0 3it 1 0 9 1017 '1 • 1 0 1 9Iti 1. 0 0 1J-9 1 Ù 0 12 0 L 0 0 12 1 1 3 0 42 2 0 3 0 52 3 1 0 0 52/1 I) 3 52 9 0 0 22f; I 0 0 12 1 I 0 1 2bb ■''! 4 11.-’V () 2 ,■> 930 0 2 4

31 1 0 2 332 {) 3 533 L 0 0 1

2 6 4

API EN DIX \

(iii ) TEAT J)ATA - i )KT- \I7

N U M B E R NUMBERFROND

NUMBER F’ OR ROUND

SUBSURVEY OF b/lO /77

N U M B E R T O T A L N U M B E RRICE C R A I N L E S I O N S

J -'l 1 0 30 21

4 0 13 16

3a 0 3

3 / 3 3 8

38 I I' 0 1

33 0 0 2

■1 0 J. 0 0 1

4 I 0 4 1 3

45 0 4 8 12

43 0 0 3 5

44 0 0 2

4 3 1 J 1 4

■'1 b 4 0 0 4

4 ( 0 0 1 1

4 8 0 0 2

4 ; 1 0 0 1

30 L 0 8 9

31 s 0 ) 5

3/-' 0 4 6

33 0 4 6 10

34 0 0 10 10

33 1 1 3 4

36 4 0 3 7

3 7 0 0 2 2

3 8 1 0 1 2

33 L 0 0 1

a 0 i 0 0 1

o L 1 0 0 1

bU 0 0 I 1

b 3 0 0 2

0/1 0 0 2

b'y 0 L 0 1

O O 0 ' ' 3 9

265

APPENDIX

) T EAT _DATA„..:i.444TAli- 8 U B J U R V E Y OF b/lO/77

U M B E R N U M B E R N U M B E R i'9,a N U M B E R T O T A L N U M B E RF R O N D a 2 R O ’ND Kiel U R AIN L E S I O N S

67 1 0 0 16 8 0 1. 4 96 9 0 0 13 1370 0 0 571 0 0 2

71" 0 3 0 373 3 0 0 574 1 0 0 173 1 0 0 176 3 0 1 4' ( ' ( L 3 6

78 0 : ' 4 6

79 1 1 6 8bU 1 v) 0 1bl J. 0 0 1b.: 0 i 0 1

8 3 0 L 0 1

8 4 0 1 0 1ü'j / 0 0 2

bf) 1 0 0 1

8 7 1 0 0 188 1 0 0 1

89 0 5 6 890 2 3 6 1191 1 0 3 4u , ' .1 0 0 19 3 I 0 0 1

94 3 0 3 6

9 9 0 49 6 0 2 49 7 0 0 2

9 b 0 1 0 1

266

A P P E N D I X B - Me u;u fvvnen t o i BPV particle d Lame le ro .E X T R A C T F‘ROM H C A (Tent Rice G iriir - cingle case)

P ar licit) N u m b V .1'

M c A

■aen reme i)

n IV X ;n ) P 11-1 i c ), e M e a. ou rem en t ( mm) M 'uii Ku.aber A B M e a n

1 4 . 84 . 9 4. 3

'). 193 4.6 4.0 4.9 4. P| 8 4 4 . 2 4. 2 4 .2

)4

/}. 1 4 . a

■'.9■ j. 0

4 . 0 23 4 . 9 4 . 4 4 . 9 9/)

9 1 • ' 1. 0 4.1I; 4.0 ) . 8 3. 9( ) . a -4 . 0 3 . 8

s 3.8 4. 4 . 09 3.8 4.4 3, . I

10 3.8 3.8 3.811 9.9 4. 9 4 . P-

I P 3.8 4.'-' /]. 013 3 . I-' 4.0 9.814 3.0 3 . 8 3. ' (

19 3.8 3.9 . ü 9lo ;. 9 '1. 0 ■j. ' ' '■ '1/ 4.8

,1 1 ,

1 . 3 4 . 4

19' 1 . '

3.n1

' I .

j 1

3 . 8 8

' ' 0 p I

1 • '

3.8t . a

'1 . 01. '

9 . 93 . 8 P|. 0 J. 9

A B " twh .,1 i o n u ' l e f iiu-e eu n-laei i I e made a t 9 0 °

t o oac i i o L l i e i ' .

2Ü7

API'EN DIX B E X T R A C T P R O

P a r L j. c 1 o Numb e r

.Mf.iüU l'.ya'M t c ! UPV p a r t i r Le d lame t e ro .

R 0 R (Te.'it Rica,- C rain - S ingle case)

Mt'asu L'cmen 1, ( mrn ,i A B M o 1. -, ; t

rnrticj.e M e a s u r e m e n t (mm) Nni:ioer A B M e a n

1 4 0 4. 3 4 18 4 l 4.1 4 1

2 /i 0 J.31 3 ' ) \ j 24 3 0 4.5 4 23 3 ■/o 4. 1 3> 4' 3 2 8 3 7 4.1 3 94 3 8 3. ' ) 3 8') i ’ O 4 0 4.1 4 053 3 pi ' t ' > • 7 4 2 3 . 8 4 .00(; P| 0 8 ') 8 2 8 4 1 4 .0 2|. 05'/' 4 ■ •0 ,1 18 2 ■ ' 2| 0 3 . 8 3 Cl8 3 S ' 1 8 0 30 4 0 4.0 4 0

M 3 / 3l 4 0 4.0 4 010 4 0 J.0 3 2 8 3‘ '1 1 3 . 9 4 0

11 > 0 4 •R) 3 l8 3 > 4 0 5.9 3 95

1. 1' \ - 4 ■0 i 1 34 3 8 2| . 0 3 9013 " 1. 1 ■1 18 38 4 2 3.9 4 0514 4 o / | . 0 4 3 38 3. o 4 . 0 3. 95

i'3 4. /i 1 L 4 2 3' 37 4 0 3 . 8 3. 91'. /( '' ' i , ' ) 36' 4 1 3.9 4. 0

1/ 4 - 4. 1 4 l 3 30 3. a 4. 2 4. 018 4. ] 3. ( t 4 0 4 0 4 0 3 . 8 3. 9

10 4 1 1.J. 4 .1 41 3 h 3.9 3. 75

2 0 4. 0 4. 4 1 42 3 0 3. 8 3. 8521 4 1 ' i. '1 4 3 3 9 3 . 8 3 85

6' 0 4. 0 3 0 ‘) 44 4 0 4 . 1 4. 05

A and B - i \\l‘ (11 nnie1 ' '‘ lin CI t in- rr ie i i t; mad e il 9 0 °

t e C' a c 11 c l ! n ■ 1 \

2 6 8

A P P E N D I X B -

E X T R A C T F R O M

Par tic le Nu iûb er

:e ■ ,iu î v II t P :' PV joa r 11 c 1 c; d L am e b0 rs , R C (Teat Rlct' Grain - S ingle case)

Meaau r e m a n m m ) A B M ean

Par t i d e N un Poe r

M e a s u r e m e n t (mm) A B Mean

1 3.9 J. 9 3.9 2 3 3. 9 3 . 9 3. 9

p 3.B 4 .. ' /|. 0 2 4 4 . 0 3 . 8 3 . 9

3 4 . 0 /J . 0 4. 0 29 3 . 8 4 . 2 4 . 0

4 4. < -1.19 2o 9. 9 4 . 1 4 . 0

5 3.9 ■3. '3 3. '3 2 7 4 . 2 4 . 5 4 . 3 36 3.B .1. 0 3.9 2d 4. 2 4 . 1 4 . 1 5

7 0 4.93 -'9 4 • 9 4 . 8 4 . 6 9

b 4. 4.28 30 4 . 2 4 . 1 4 . 1 5

y 4 . 3 1.2 4. 32 31 4 . 7 4 . 5 4 . 610 4.6 -1 . ' ' 32 3 . 9 4 . 0 3 . 9 511 4. 0 4.1 4 . O' - 3 3 4. 0 3 . 9 3 . 9 3IP .'1.2 ).■' 4 . 3 ' ' 34 3.8 3 . 8 3 . 8

13 /'1.9 '.. u 4.9 2 3') 3.9 4 . 0 3 . 9 3

1-1 ,. 0 . o 4 . -i 3" 3 . 8 4 . 0 3 . 9

13 4.P 4. ; 4 . 32.; 3/ 3. ■ ■/ 3 . 9 3 . 8

I' 4.. ' ) • ' 1 . 3 ' 98 3 . 9 3 . 9 3 . 9

ll 4 .1 4. 3 39 3.9 3 . 7 3 . 8

IB 4 . ■' 4 ..'! 40 4.0 3. 9 3 . 9 3

19 4.1 4.1 '!. 1 41 3 . 9 3 . 9 3 . 9

2 0 4. -1 . . ’ 3 42 3 . 7 4 . 0 3.8321 4.2 4.1 4.3 43 3 . 9 4 . 0 3 . 9 3

2 2 4 . 0 3.7 3 .89 4 4 4 . 0 4 . 0 4 . 0

A and B - t VV 0 Jia.me to; measurements made at

9 0° to vaicl i o t h e r .

269

d'UCNDIX B - K e isu rame cl el 9 2V aar 11 clo d i am e 1 è r e .

T R A C T F R O .1 ( h ( i e -, t H i e c i4 rain - S ingle cose)

hir tic 1 e ;2 e i. s : ! r e .111 ' a t ( mm ) 2 a "'t i c Lc M e a s u r e m e n t ( mm)u m b e r /I 1' 10, a a •) umue r A B Me an

1 3 . 9 '1. 0 3 . 9 9 93 3.8 3.9 3 85

2 4 . 2 , a .'1 . u , ' 3. 8 3.0 3 85

3 4 . . 4 . 0 4 . 1 29 3.B 3 . 6 3 74 4. 0 4. a 3. 9 ■ u 9.8 3.9 3 85

3 3. 7 '■. a 74, 9 /' 4 . 9 4.0 4 25u i a) d . o ■3.'2„, a a 3.9 4.0 3 95

7 3.9' 4 . 0 :').9 9 22 3.9 3.9 3 9a 4 . 6 ■I. 1 ;. a 9 40 3.8 4.9 3 85

9 . 2) -.9 ■J. a ' J 4, L 3 . 8 3.8 3 a

10 4 « 0 I. L d • i- ' 3. 8 3 . 9 3 85

11 3. 9 Î. 1- 4. 0 4 3 3 . 7 3 « 8' 3 75

1, 4 . a 1.0 3, '4 9 4 3.8 3.9 3 85

13 4 . ; 4 . 4 . 09 9 9 9.8 4.1 3 9 514 4. a 1.0 4 . 4’ 3') 4.0 3.9 3 95

19 4 . O' d » 4.1 3/ 3.9 3.9 3 9lo 3.4' a 9.69 4 a 4.3 ■1. 7 4 5

17 4. a, 4 . a 4 . ar 39 ■-1. 9 4 . 2 4 35

16 4. a 4 . a 0 . a 40 4.2 4 . 9 4 35

19 3 . 4) 4 . a 3. a r 41 4 . 9 4 . 0 4 252 0 4 . 2 4'. 9 4. 04 ' t ‘ ■ 4 . 5 4.2 4 3521 /| . 0 -I . 0 4.0 3. 9 3 952 2 3 . B 4 . 0 3.3' 44 4 . 9 4 . 3 4 . 4

A and I/,

VO^ L o f 1 1 u Id i r r .

d i.' i iüh l.h r nu a ;ai IV i:n ai I, s made a t

70

A P P E N D I X B - Me ■.Virement oi' Bi'V par'tide d i ameters. e x t ract P'RC'M H I ] (Tv; 11 Rice C fair - Single case)

P a r t i c l e icasu remenei mm ) i.irticie M e a s u r e m e n t (mm) N umb er A - m-.h'in Hu m o e r A B M e a n

1 4.2 Op 4.1 9.8 3.95■;/ ■! . ' 24 4 . 0 9.1) 9.89 4 . 0 'U ■ 4.1 2 0 4.9' 9.8 4,154 ,'i. ■ ' 4. Oo o4j 4. 0 9.8 9.95 . a 0.9 3. Bp 27 4.9 9.9 4.1b 4 V 2 /] . 0 i. 1 2 B 4.1 4.0 4.057 4. 0 4 . 1 4 . O'; .03 9.9 4.0 4.05a 9.9 -1. 0 3.9 9 30 9.9 4 . 0 9.959 >. ' > 3. a 3. ( 31 4.1 9.9 4.0

10 9 • 9 . ' ) * / 32 4.1 4.0 4.0511 3.6 ,. o' 3. B 3 3 4. 3 4.2 4.251:' 4 . 0 3.H j . 0 34 9.9 3.8 9 .8 5

13 3. H ■■ . H 3. B -2> 4.0 9.9 9 .95

14 3. ' ' . B B2 3': 4. 0 9.8 9.919 3. 3 4. 0 3 . 9 4 37 4.0 4.0 4.0Lo H '■. 1 3B 4. 0 9.9 9 .95

i'/ 3 . 6 1.0 3'. ' ) 30 4.0 4 . 2 4.1LB 3 . " . B . i 4 0 9. B ■1.1 9 .95

19 '1 . 1 ■ 9.0 4. 0 41 9.9 9.9 9.92 0 3.9 3.7 p . B 4 2 9. B 4.2 4,021 4.2 3.B 4 . 0 4 3 4.9 9.7 4.12 2 4.2 3. B 4. 0 4'i 9.8 9.9 9 . 85

A and B - I VJ 0 dioiiu, to 10 measuL'omenti3 mad e at

90° to o;u;l! olhor.

2 7 1

APl-'ENDlX 3 - Me ,;.ju rvmcii !, ji 31’'/ p a r l i d c diametorn. E X T R A C T E R C M R C P (Teat Rice Cra i n - Si n g l e case)

P a r t i d e Number

M e asu rcmmi t ( mm ) A 3 Mccn.

P a r t i c l e R umber

M e a s u r e m e n t (mm) A B Mean

1 4.2 4 .0 i d 4 9 9.9 9.9 9.9

i.'i O 0/I. ( '

. ' j 2| . 1

:. ', , . 1.2,

2 4 4 d 5.9 4.0

/ ' ( . 1

o 4 . 0 A , 1Y ,. i ;. 1 4.1Ü . 8 9.89 ;.8 i. 0 .9 . ■'

10 5 . 9 5. d d 11 L 5. 1.0 d . 81.' 4. 0 ' i ).919 9.9 d. 9 9.d)14 4.- 4. 0Id 4.0 . 8 9.9l o 4.2 i. 1 .1. 117 ■i. 1 ■1, ( ) /1. 018 4.0 /]. 019 . 9 4 .1 4.0PO d 9 9.8 9.8d 9. '( 9.8 9.7P p /'1. 1 4 . 1 /'I , 1

A and B L iV 11 iaiML 1a L '40 to 1 ' a c 11 • ; IP o r

272

A P P E N D I X P - Mo Oju mnaril ei' iPV partiel» d 1am e t era.EXTRACT PROM R : C (To t, Rice C r a i n - poolo J )

P a r t i c l e N umb er

;/i r a L .A

o rumen I P ÎV

I ),j an

Partic j t, N urnber

3 iVI e a s A

u renient (mir B M e a n

L 9 . 0 >. 1 4 . 8 4.9 4.894 .0 >. 0 - ' ' i 4.9 9 . 0 9.09

9 d.o 9.1, ‘ ). 1 9. 0 9.09'1 9. 0 o. 0 P O' 9.0 /]. 0 4.8J 9.0 ', 1 9.0 4.7 4 . 8 5

u 9. O' 4 . 7 4.89 P8 4 . 8 4 . 8 4 . 8

■/ 9 . 0 p. L p. 09 PO 9.1 5.2 5.158 4.8 0. ), 1 p 90 9 o 1 5.1 5.1

4 d 1. - . 0 91 9.2 5. 5 5 .25

10 . 0 0.0 o . 1 9-' 9.1 9. 9 9.211 4. 1 / 't. 9 1. 8 99 9.0 5.1 5 .05IP 9 . 0 '. 1 9 94 9.9 9.0 5.2519 9.8 0 . 1 OP 9') 9 . 2 9. 0 9.414 d. ■ 9') 4 .8 4o9 4 . 8 5

19 4. ') O'. 0 1.06 97 4.8 4.9 4 . 8 5lo 4. P . 0 1 . 0-. 98 4 . 7 9 . 0 4.8517 4 . >. 0 4.5 4. c 4.5918 9 1 ' ' 0.0 J. ’ a ■40 4.7 4.8 4.7510 4 . 8 ' . 0 4.2 ■11 4 . o 9 . 2 4.9P 0 4 . 8 4 1 . 8 4 ' 4 . 9 9.1 5.0P I 4 . '/ 9 . 0 1. 8 9 4 9 9.9 4.7 5.12 4. 9 9. 0 4.79 4 4 4.8 5.0 9.2

A and B - two tj i a rn u t r i[iea,sureinents madeci. 'b 9 0 ° l o each other

273

APi'i NDIX B - KX'I'RACT f'RuA

Par t i d e H u m b e r

;v'e u II t o i ' . r i r i l e L e d t a m e t e r e ,

!i a ( T c - i i . B i e r d r e L ü - p o o l e d )

'■'.eii.eu roîp.en It in.il) i or t i d e Ueaeuremeri t ( mm) B M ean Nii.'iiDer A. B M e a n

1 d . 4 4 1 'b o 0 ' ) . 0 4 . 0 4 . 0

d / 4 . 1 4 4 4 . 4 4 . 0 4 . 1 4

4 . 0 1 t ) . 04 4 4 4 . Y '1. 9 4 . 8

4 4.1 ■) 4 . ' ' :o) 4 . 0 4 . 9 4 . 9 4

4 4 . 0 B B B . 1 4 4 Y 4 . 4 4 . 1 4 . 0

6 4 . 4 L B . O 4 8 4 . 1 4 . 7 4 . 9

Y 0. B . ■ .'B 4 . 9 4 . 8 4 . 8 4

B / | . Y V B . , ■ 40 4 . 4 4 . 8 4 . 6 4

4 4 . B 1 B 4. o ' 41 4 . 9 4.4' 4 . 0 4

10 . ' B 44 4. 0 4 . 1 4 . 0 4

11 B . t ', . /| 4.4 4 . 0 4 . 1

1. i . ' ' 44 4 . 4 4 . 1 4 . 4

11 .'1 . < ■■} i ■1 . VB 44 / | . B 4 . 0 4 . 9

1 1 Bj.H L 4 . 41 4’ ' 4 . .1. 4 . 4 4 . 4

I t ' ' 1 .0 '1 0 . i . B ) I 4. Y 4 . 0 4. 84

l e If . i : ■; i . 0 ;B 4 . 0 4 . 1 4 . 1

I V B . O B, i B ' ) 4 . 1 4 . 1 4 . 1 4

1 B d . o i. ■i.4'> 4;0 ‘1 . 9 4. :3 4 . 1

14 4 . 0 /| : 1 ' 1 .44 41 4 . 0 4 . 1 4 . 1

1 0 /I . 4 t> 1 ‘ ; . 0 /|4 4 . 9 4 . 1 4.04'1 y. 1 ■1 4 4 . 0 4 . 1 4 . 0 4

11 4 . 4 ll to 44 4 4 4 . 1 4 . 1 4 . 2

A ni i i i B t vv o d 1.am'.OaM' meanut e m e n t s mad e

a t 4(7 ' t o o :. c }[ 0 L ! 1 e n .

2 7 4

AJO''E!'î,DTX B - Me.-ôu m'ijon t b i ’ V p arLicie d l anetero.

E X T R A C T F R O M H C C (Tuat Rice Gra i n - pooled)

Par 1 i, c .1 e M e a s a reiiaui . V iiîm 1N u m b e r A 1' P'an

i B . M 0 4. 9/la* tu B B . i.

.B/]

. ’ ' ' . L

'i • / B . ' '

1 . C) B

B| . ) t> . 0 4.1B

.1 . 't ‘ . B . i-

7 B . , B . O ;. 1y B-. . 0

V B.O Bu. 7 4.^'i10 4.0 a O •;.9 911 4 . 9 3'. 1 0.0

1,: 4 . V ■1.90

ParlLc i e K mil De r

M e a s u r e m e n t (mm) A B M e a n

275

Ai MOON DIX B - Me .7i U r''iB'nnl .21 Mi'T pa r lie; ] e d i ameters.E X T R A C T P R O M !■’A (Te a L i' rend - n i rir;P '' case)

Par Liei e M e :.-;ula.anen tt 'iim ) Idirticle Me a sûre m e n t (muI'iunib er A 8 N u a b 0 !■ A B M e a n

1 4. 0 4 . 4 4 . 2 2) 4.0 4.8 4.91 4 .4 • ■ l.Uv ■ ’ 'i 4 . 4 4.9 4.74 4. 4 « 4 4 . 7 21 4. 8 1 . 1 1.15

4 4.8 4 . 8 4u8 2fc 4.0 5. 3 5 .15

4 4. 2 4 .7 4. o 2 7 4.0 5.2 1.16 4 . 4 ■.)• ■ - 4 - 8 4 28 4.8 4 . 8 4,87 4.'-' . ( 4 • 8 2; 21' 4.2 4.9 4 .558 4. 8 4 . 7 4. 74 40 4. 8 4 o 3 4 .55n . 1. ' 1 ■ ‘. ■) 4. I 41 4.5 4.7 4.5

1 0 /{. ' 1 4 , 8 4 .44 42 4 . 2) 1 . 0 4 . 8

11 4 . t 8 • 1.0 4 3 4 .2 4 . 8 ,4.11/ 4.0 ' 4,1 . 44 1.2 1 . 0 1.11) ■ ' . u 1 i . 1 ' 7 ' / 4.9 1.0 4.9114 . (.' '« ■ 4. L 4- . 0 4.9 4 .95

11 2' • ' 1 . ( 1 4 . 1 4 7 2). 0 4.9 4 .951(; 2 . 0 4 • ' 2, . 1 2 8 1.2 4 . 9 5.01L7 0 . 0 2). O' 42) 1 . 3 1.0 1.1118 2; . 0 4 .4 1.4 2> 4 0 4.0 1.1 1.0514 -1 . 8 4 . 0 4 . " 41 1.2 1.9 1 .5520 4 . 8 /l . 8 4 , 8 41 4.9 5.0 4 .95b 1 /|. 4 . 0 4.4 2 '14 1.1 1 , 0 4.052 2 4. 8 1 . 0 4 , 4' 4 4 1 , 0 5.1

A and 8 tv/o d i niiir- 1 e r measLi remerits made at4 0 ° U) (!':iO ll () t he r ,

!76

Ai- ' ] - 'Ei IDIX B Me .c j t : î s m i c n l o'.' iM’V p a n i o l o d i a m e t e r s .

EXTRACT FROM FA (To : i ,b i ' r u n . i n . l n p ; ! ' - c a . i o )

P a r L ie l e Me a.; a roi i ien m.i; ) Pa r t i c iHumb e r A ;J IB aaa. Nuiabei*

M o a s u r e m e n t (mm) A B Mean

. 04 . 8

1 . 0

1 , 0

. 8

) . i

1 . 0 4. 1

. r

A a .n l B - t e a r l i / u i r . ta r m e a s u r e m e n t e

m; .da a t 4 0 t o . ' . •u l i a t l i c - r .

!77

APi’ENDIX B Mo, 1 su rouie n I u ï lil’V purtlcie d i a m e t e r E X T R A C T FRO.!' l'Ri - (sLi.p.'lo c;j:u' Loai, L'rond)

P a r t i c l e Humb o r

,\1e a su i-’u.'ten t( mm ) A B Moan

A and . 0

''‘.■i.i'i, i c le t| u:a t> o r


1 4.8 4.1 4. 91 9 4-. 1 4. o 4.114, 1 1.9 4. 0 8 4 4-. à 4. 0 4.9

3 4.1 '1. .1 4.41 .'-'1 4.8 1.0 4. 6-I 4 . ' '■I. ■1.4 8'-. 4.8 4.9 4.819 1.0 1,0 d . 0 8 7 1.0 1.0 1.0d 1.0 4. ' 4.91 ,:J 8 4. 8 4.7 4.71/' /; . 8 1 • .'1 . Od '29 4 . 8 4.1 4.65u 1.0 4 . 8 4.0 90 8| . 1 4.6 4.119 4. ■ 4.1 91 1.0 1. 8 1.1

10 ' ' i » 4. 0 9' ' /|. 8 1. 9 1.05il '1 . 1| . 0 0 « I 9 3 .'1. 8 1.0 4.9Id ' i • j . ■ 4. ' ■34 1.0 1.0 1.019 4. 8 1.7 i|. /I 92» 1. I 1.0 1.0511 V. ! I ' . O l 9n 4.1 4.8 4.65L 1 1. ' ■ I . 8 2’ 9 7 4.7 4. B 4 .75I d 4. 4.0 4 . d 98 4.1 4. /' 4.617 4.9 4 u ' 99 1.8 1.1 1.9518 •1. 0 1.0 /i. 4 0 lu 9 4.9 1.119 4.0 4. ■; 4 . 4 1 41 1.1 8.0 5025.8 0 4.1 4,1 4. 21 4.' 4 , 8 4 . 8 4.881 4.7 4. ■' 4.1 4 9 ' ) . 0 1.8 1.18 8 4.1 1.0 4.8' 44 1.1 1.1 1.1

t w a ( 1 i ; un ( ' t i 1 r' m (\'i ru r ir. o n t s nu-i d e a t90 to -rich other.

2 7 8

APPENDIX B - t.ïLd'S'u ra inen t c I [D'Y r a r i i c i o d i a m e t e r s .

EXTRACT PROM PB ( E l i v ï I. o es, s c l a a t lO 'cnd )

Part i, c i. e

N u m b e r(/asu riaueti tt Uïm ;

;j Ma-UlP .'I . I. t j . c i O

R Lim b e rM e a s u r e m e n t (mm) A 3 M e a n

1 4 . 8 ,. B '. 9 ' ;3 1.8 lui 1.918 2> . 1 4.8. 4.0 8 4 1. d' 4.1 1.0

9 1.0 .'1 , B ■1 . 8 : d 7;. 0 4. ') 1.914 1.0 J * ' d. 1 8 C 4. a 1.2 1.0

1 4.8 .0 ■4 ■ ' ' 4. a 1.8 1.0b 8 . 0 8i . B 4.8 8 0 1. 8 1. 1 1.11'( ' ). 'H 4. d 8.1 1.018 d . d. L 90 7'. 0 1. 3 1.119 /i . 8 I.d i . Od 9 L 4.8 1. 1.0

J-0 4.'- 4.8 4 » V 'd 4.7 4.1 4. 611 4. d 4 . 7 4.0 4 . 1 4.9 4.71i.: 4 . 8 i. d 94 4.1 1.1 4.819 1 . 1 d . . ’ d. Id 8d 4. 8 4.0 4.8114 1 • d' d „ ' .1 9i> 4 . 8 4.9 4.8111 1. 4 8 . 8 1 > 8 97 4.1 4.9 4.7le 4.8 . 9 ‘ . L 90 4.8 1.0 4.917 1. ’ d ■? ' 973 4. 8 4.9 4.81

IB ) * < - 8;.- ■ 1.0 7' 40 4.1 4. 8 4.61

19 4.7 ■1.7 41 4. 8 4.8 4.88 0 4 .0 !. 0 21 .9 4 ■ ' 4.1 4.8 4 . 6 5

81 4.8 ' » ' i ■ 05 4 '3 4.8 4.9 4.818 8 4. d 4. ■ 44 1.0 1.0 1.0

A diuJ B l w t, d i. ame 1 Cr ■ uloasu ren ie n t s mad. es,t 90 le es.ch („>LLor.

279

APPENDIX 3 ~ MtU:3u r-cment o f Bl'V p a r t i o i e d i a m e t e r s .

EXTRACT FROM EB ( E Lnp;i , ' ca,;u; Lea L P ret id )

NumberP a r t i c l e Measu j 'emt;n I t mm ) P a r t i e 1 e M e a s u r e m e n t (ram)

Mean N amber Mean

1 1 . 8 9 . 0 4.9' 4, a 4 . 6 5

- 9 . 0 4.B9 4-1 4 . 9 / | . 8 4 . 8 5

9 9 • ' ' . o a. 19' 9 9 9. 9 4 . 9 9 . 1

4 4 . L 4 . { 4 . 7' , ' b 9 . 9 9 . 0 9 . 1 5

1 4. ) 9*9 4*79 .'7 4 * 7 4*9 4 . 8

b 2| . 7 9 .L 4 . 9' , ' B 4 * 9 4 * 9 4 . 7

Y 9*9 9 . 0 ' . 1 '-'9 4. B 9 . 0 4 . 9

B 9 * 9 4* 9 . 0 90 4 * 9 4* 9 4 . 9

9 4* { a. 9 . 0 91 4 . 8 5 , 0 4 . 9

10 4 . n 4 . 9 9| . ’('J 9'-' 9 . 0 9 . 0 9 . 0

Li 4 . 9 4. Y 4 . 1' 99 4 * 7 4* 9 4 . 8

19 4 * 7' 4 • ' ; 94 9 . 1 9 . 9 9 . 2

13 4 . ); a . 0 4, ^ 49 4 . B 9 . 0 4 . 9

1/1 a • 4. ' /| * 7 4') 4 . B 4 . 9 4 . 8 9

19 4 . ( a . 9 4 . B 9 :// /]. y 9 . 0 4 . 9

l a 4. 9 a . 1

1 / 4 . B 9 , 9 9 . 0

I B '1.7) 9 . 7 9. L

19 4 . B 4.B I .B

9 0 4 .7' 4 . 9 4 , B91 4 . B 4 . B 4 . B9 y' 4 . B 4 . 9 ' 1, B9

A and B t W C; d lame te v jrieasu taniientsmade at 9 0 ° to each oLhcm;

280

A P P E N D I X B - Me,

E X T R A C T F ROM [c

a;u t'. 'inc

9 (T(m l oi' npv

;a I I'fmi'i

j)a r 11 c I e

- pooled

d t amelers. s a m p l e )

P a r l i c i e M u ;isu romt n I ( m.n ) Par Iic io Me as u r e m e n t ( mm)N u m b e r A B M o a n Nu m b e r A B M e a n

1 4 . d 8 . 0 4 . 9 8 3 4.8 5.4 5. 1

4 . d 8, . 9 4 . 8') 84 4 . 8 9.9 9.19 9, 9 9 . 0 8 5 4 . 8 5.0 4.9/] 4.9 1 9.0 8 6 9.1 9. 9 5.29 9.0 9.0 9.0 8 7 4. 8 4.9 4.850 4 . d 9 . ' ' ;.19 88 4 . b 4.9 4.79Y 9.0 ' * ■ 9 . 1 89 4 . 9 9.8 5.29y /] . 8 'a 0 4 . 9 90 9.0 4 . 6 4.89 4 . 9 9. 0 4.9'» 91 9.0 9.0 9. 0

10 4 . ij 8.0 4 . 8 9') 4 . 8 6.0 5.4L I 4. H /| . 8 8 59 9 .9 9.2 9.9918 4 . 4 ' . V ' 4. 1 5'1 9.0 9 . 2 9 .11 :» /]. ’ ' ' i • 4 .' 98 b . 0 b , 0 6 . 0

1/| 4 . 8 1 ■ '9 9" 4.9 9 . 0 4.7919 /I. d ■ '. 1. 4 .'O' 9 V 9.0 9.0 5.0lo i. 0 1 . 8 4 . 8 98 8.0 9 . 8 9.1IV 9 . 0 9 . ,. 1 99 9 . 0 9.7 9. 99

Id 4 . 9 8|. J ■4 .8 8' 4 0 9.9 9 . 8 9.2919 4 . 8 /] . ■ /] . H ‘; 41 5.0 5.4 5.2

8 0 4.8 /; . 'V ■i . ' ' 48 4 . 9 9. 2 4.8581 4.8 4 9 4.9 5.0 4.958:’ 4 . 0 9 . 0 4 . 8 44 4.9 9.0 4o 95

A and H1 - L w r. d i Mini'It.' r meamn remt ; n t s made

a 1. 90» Lo eac h otlior.

2 8 1

A Pi'EN 1)1A B — M e ! s'l I ' . ' .leat 0 1’ K87 pa r L i 0 1 e d 1 ame te ro ,E X T R A C T FRO..: 14 C (T.'■it, I'rorid, pooled 8 a,m pie )

P a. r I i c 1 e M e j r>il rei.'leri 11 m;n ) P a r t i c le M e a s û r e m e n t (ram)N umb er A I) 91i>an ï’i nmbe r A B M e a n

1 /|, 9 9'. 1. ' ) n 0 83 4.8 5.0 4 . 9

9.0 9. 9.1': ' » -1 ■ - • 1 4 . 0 4 . 5 4 . 2 5

9 4.9' 9. . 0 '1 .79 4.9 4 . 9 4.74 4. : ' ■1. 0 4.8 4.4' j 4 . ■/ 4 . B ■ } * f ' ■"1 '1. 9' 5 . 0 5 . 0 5

u. 4 . B ' ! . /' '■.0) B 4 . 0 4 . 0 4 . 0

7 4 » J * ■ 4. B9 99 4 . 8 4.5 4 . 5 5

B 4. . 1 . 9 |.9‘. 40 1.9 4 . b 4 . 4

4.'' (8 4 . 7 9 91 4 . 8 4 . 9 4 . 5 5

iO ■ 1 • ; ■ . (' 1 . 'V9'11 y.l 4 . B '1. 8 ;18 4. B 4. B 4.,B19 4.8 ». 9 .'10 B ' )14 4 . ’' '1 . 8 'Î. 718; 4 . B 4. 9In 9.0 4.7 8| . B917 9o 3 ' , 9 9 . 1 ’,)LB 4u8 9.0 9;. V 919 4. 9 9 . 0 .'1,9 9.'(.) 4 . 9 ' 4 , B9

81 /'1.7 9.9 4 . 0 9

8 4. ( 9. n 9.19

A and h L v'j o d isMii ■ 7. c- r irieasu re men tam.-iclc* ;i I, ‘ 8 o CMid; oi .hcf

282

APl’E N D I X 13 -• Me iSLi r.aneiit, Oi' i'n'V partic.ie d i ameters, E X T R A C T P R O M P d ( (loi) Lo ; osmplo, to.at fiat anci round)

article Me;asu roiru'ïi I ( mm ) L’ar l i c L e Mes..sûrement (mi!umbo r A y ; ' - a 11 N u .n b n r A B Mean

1 0 t . B /I o 9' , ■’ ■; 4. y 4.8 4 . 7

'i . 9 4.9 4. 79 . 4 4 . 8 4 . 9 4 . 6 5

5 . 1 4 . 9 9 89 5 . 0 4 . 9 4 . 9 5

4 . ’ 0 5 . 9 5 . 9 5 . 9

5. 0 4 . y 4.9) ( 5 . 9 5 . 9 5 . 9

0 4.7 4. y 4.7') . ’ y 5 . 8 4 . 0 3 . 9

Y 4. /. 0 4 . 7 9 5 . 7 4 . 8 5 . 9 5

y 4.9 0 5 . 9 4.1 4 , 0

77 , 0 4.'-' 1. ') ' ' 51 5 . 9 4. 4 4 . 1 5

10 . 1 4 . B 4 . 9 9 4 . 5 4.0 4 . 2 5

11 9 . 0 ! .9 /]. 9 53 4 . 5 5 . 9 4 . 1

18 ' /. 0 9[ . 8. .1 , 9) 54 4.. I 5 . 7 5.914 4. ■ ■1. y ■ 1 P / 3 3» 4 . 1 4. 5 4 . 2

14 'I, y I . o ' i * / 99 4 . Ü 4 . 1 4 . 0 5

15 5 . 0 4 .9 4 . 9 57 4 . 0 4 . 2 4 . 1

lb 9.0 /1.7 4 . 09' 5B 4 • 5 4 . 2 4 . 2 5

1 / 9. 0 4 .9 4.9 9 59 4. 5 4 , 2 4 . 2 5

ly 4 . B 4." 4.B5 4 0 5 . 8 4 . ..t 4 . 0

19 9.0 4 . 9* 4 . 9 5 41 4 .1 4 . 2 4 . 1 5

8 0 5.0 4.9 4 . y 4:' 5 . 8 4 . 0 3 . 9

8 1 5.1 4. y 4 . 9 5 4 5 5 . 9 4 . 1 4 . 08 8 4u9 4. i 4 0 9 44 4 . 1 4 . 2 4 . 1 5

A and d ■■ i: w 0 1 ila mo l.('• r me:'o; urement s madeat, 90° to o;i c; h 0 Uî e r .

283

A P P E N D I X B - Y:-:. . . j U f , '."I r;'C oD 'E’V iu'irliclo d i ameters, EXTRACT FH0..1 i»’ (poo, l.oC o.smplo tost. ils.I and. round)

P a r t i e l e Number

.Voa.:ia ro-ifH.'ii i t ii.un j r t, i c 1 e Measurement , (mm)A P iMvoii't Nu-iib(.'r A B Mean

1 5.9 4 . 9' .92 4 . 9 9 . 0 4.794 . 0 /| , t ■' ■1, 0 24 4. 7>. 3 4.9

5 9 . 9 ,0 H 9 . B' 29 4 . 7.) 4.9 4.73 .9 4 . 9 4 . i. I ’ u 4. 7 4.8 4.79

9 3. 8 4u 't , 0 ' 27 4 . ‘3 4.9 4.90 4 . ' : . 0 ‘■'1, O'. ; ' tt 4 . 0 4.9 4.257 3 . 0 . 0 . 0 ,3 ' j /I 3 4 . 9 4.4B. 4 . 3 i . 9 4 39 5 . 0 4 . 2 4 . 6

9 4 " ' /. 0 ■ t ' ( 2 31 4 . 0 9 . 2 4. 6

10 4.2 2, • 1 32 4 . 1 4.2 4.1911 ,1. 0 4 . B 33 4. 3> 4 .2 4 . 39

18 4 . 2 9 . 0 4 34 4. 2 9 . 0 4 . 6

15 4. 0 ' , u 4 « 39 4 . 3 4.0 4.2514 .3.9 4,9 4 .4 3o 4. 3 4 . 6 4.9919 4 . ' 9 . 9 2| . 9' 3'V 4 9 4 . 7 4 . 6

19 4.9 ' ' . ■1.9' ; 3 9 4 0 4.9 4.4517 4 . 9 9 . . 8 3» 39 4 7 4 . 8 4.75l o 4.5 4 . 8 4 . 4 40 4 . 9 4 . 8 4.6919 4 . 4 . 8 41 4 7 5. 2 4.992 0 4 .1 4 .. ’ ■1. 17; 42 4 . 2 4 . 7 4.4521 2, . 9 4 . ( 4 3» 4 6 /). (» 4 . 6

2 2 4.9 4. '/ . 9 4 4 4 3 9 . 1 4.7

A a n d P, - I w 0 d i a m 11 r 0 m e a s u r e men I s mad e

a t 90 t o o.s.oii (.)t.i ;o! ' .

235

.Pp ENDIX î7 M 0 ,1Hn T' bl- ■ til ol' 41’V par licie d 1 a-'ïi e t e r y .IXTRACT FiJOA L' 17 ( 1)00 Los 0.0 m [ill' , 'b’i. !. fin t and round, )

Par Lie le M 0 as u reiiion L ( laiii ) Part1c1e Measurement (mm)Nufjib or A B Moan Numoer A B Mean

.1. 4.7 1 . 8 4.7' 2 8 4.9 9 . 0 4.792 4 . 8 4.8 4.‘‘ 24 4 . 8 9.9 9 . 0

3 'i . 2 4.0 4.8b 2 b 4 .9 4 . 9 4 . 7

4 4.2 1. 7 4 . 9 4 . 8 4 . 6 5

3 4 . •: 4.d 4.2 2 ' 9 . 0 9 . 9 9 . 1

u 4 . 0 4«2 4 . 4 2 2 8, 4 . 8 5 . 0 4 . 9

/ ■1. 0 4 . 8 4 . 2i 4 9 9. b:' 3 . 0 9 . 1

8 4 . 2 ' ■ . b 4 . ,! ) 0 /|. 8 4, 8 4 . B9 4. ( ) 4P' 4 .7'' 31 4 . 8 4 . B 4.8

1 0 4.7' 8 . 8 4.82 32 9 . 0 5 . 0 9 . 0

1 1 4.8 4.8- 3 b 4 .9 5 . 0 4 . 9 9

18 4,2 2 . 0 2| , rio 3 2| 9. 3 4 . B 9.0513 2| . H 8 . 0 4 .9 32 4 . 7 4 . B 4 . 7 9

14 b . 0 36 4 . 9 9o? 9 . 0 5

1 2 4 . 8 0 , 0 8'. I 37 4 .8) ‘3.0 4.79Lb ,•] , M b . lb -, 8 /|. 9 4.9 4.917 4.9 ' . Ob ■;,o 4. 8 4 . 4 4 . 4 5

18 /:. 9 ■ ’ . 2' 2.2 4 0 4 . 0 4 . b 4o 319 4 , 9 4 . 8 i. , 4 1. 4. 3 4 . 5 4.42 0 4 .8 b . 0 4 ’ 3.9 4.1 4 . 0

21 4 .8 '. u 4 3 4.1 4 . 9 4 . 3

2 2 /|.8 ,i.L)2 4 4 4 . 5 4 . 3 4 . 4

A ■■1 ml B two 41 smote; r measurements made

at 90° to each otLier.

286

A'-’i'EMDlX B - Me.isn ïbbiieuL wi’ HPV uarliolG diaraetero, EXTR A C T F ROM F '{ (puoLod samj; I o - boa L fiat and round)

P n r t i d e Numb n r

M e a s u rrium l( mm ) A B M e a n

Particle M e a s u r e m e n t (mm) Nuiiiber A B M e a n

.L 4 . 0 d. U 2 3 4 . 0 4 . 8 4*4

2 4 » 4 4 . '» 4 . 4 2 2 4 4. 3 4 . 7 4 . 2

3 3.9 ■1. ' • 1.22 4 . 0 4 . 8 4 . 4

4. 3 .]. u. 4.4 2 2(1 4 • 3 3 . 9 4 .1

■i. t 4.2 1.22 4. 3 4 . 7 4 . 2

0 3 . 0 •;. 9 3 . B2 2 0 4. Y 2 . 1 4 . 9

/ '1 . 0 4 . 1 2 2 9 4 . 0 2 . 0 4 . 8

8 3. 9 •1. 1 1.0 3 0 4 . 0 4 . 2 4 . 2 2

') 4 . 2 ■ ! . (' 4 , ■-1 4 . 0 4 . 8 4 . 4

10 4 . ' » '! . 2| . " 2 8- 4 .0 4 . 0 4 . 0

11 / ]. 8, 4. b 4 . 2 2 4 . 2 4 .2 4 . 212 8} . 0 . ! . ■!. 1 4 .8- 4. ' ' 4 . -

1 3 » . 2 4.4 1 . Ob 3 2 4.2 4.2 4. 32

14 i . 0 t . ' I.i2 3-2 /I . ' 4.0 4 .1

12 t . 9 4. 1.2 3 I 4 .1 4 . 2 4. 3l u t . B I . . 4. 0 3B 4 .2 4 . 2 4 . 2

1 Y 4 . 8 4. 4 . 2 u 39 4.7 4 . 9 4. 8

I B 4. ' ' u . 2 2 4 0 4.U 4 .2 4 , 4

19 4.2 4 • 3 4. 4 2 41 4 .0 4 . 8 4 . 0

2 0 4. B 4. t /'1.7 4 . 8 4 . b 4 . 7

21 4. 2 4 . ■ 4 . ‘b 43 4 .0 5.9 3.9222 4. 7> 2„ 1 4. y

A and B L w ( ) uianu' Le r [liea sûrement 3 made,at 90° 0 r ■ i ( 0 o L h(' r‘.

P87

A p!-EN DIX D -

EXTRACT FROM

P a r t i e l e K umb e r

;.;s.b . e n t oP ’ i 'V sa S t i c l e d i a m e t e r s .

vVBl’V LA ( eu I a l ia o i r ; 1‘ i b l 'ü p u p i 1 i offla - s i n g l e c as e )

Ecasu ro rum t ( mm ) A il Muaii

Part ic. L e N .îiiibe r


1 '1 . 8 a . 0 .93 3 .9 4,8 4 . 0 9p 9. i a . ' 9. 8 4 4.9 4. 8 4 . 6 9

3 4 . 8 a. u a. l', 8 3 4.9 4.3 4.394 4. 7 4.0 4.89

G

7

8

9

1 0

111 2

13

14

13

l u

17

1 8

19

PO

-I . 0

- i . u

4 . 0

. 7

4 . 8.'i. 8

4 . 8

3.0

4 . 0

4.1

4. 1.

. L

4 . I

0 >

O'J

9 ’0

8

O

'I . t

A and H I wc d i a m a l a t ' maa .u,j r'onn ai t s rnacîo

a' t 9 0 L(.' l ' . ' iad ut , h e r .

20U

APl 'ENDIX B Mo l o i ; i‘u;n 'n t u i’ .'a L L o L o d i ain e l e r s .

E X T R A C T F R O M w I

Partiel- Numbur

Moaso rc-üuut A B Ü

I tu noonr i id» ru p a p i l l o m a - single case)

mm ; ut

Partie leîujiüoe-r


1 /] B 1. o ! . B a 4. 4 o8 4 a

p 4 ■; .1, ■ 4 . .-i 81 4 7 4. 7 4. 73 4 0 ’. ; 4. ' .'a 7; 0 4. y 4 9

4 4 4.0 au 3. 8 4. 1 3. 99J 4 • \ • i 4 . u ' ' / 4. 4. 4.u 4 4.18 b B 3. W 4. 4. 0i 4 B 4. 9 ■4 . B 8 8 3. 8 3. 9 3 89B 7; 3 0 4. 8,. 4 39

9 •'] i ' 1 'i • 1 bl 4. 1 4. 8 4. 4910 8: '7 4 . B ' ■j. ' 3. 6 0 4. 4il n a ., a . L ... 3. 8 4. 8 4. 3

1.; B ■ . 8 3 t 4. 0 4. a 413 ■1 B. u . 8 ;. a- 4. 8 4. b 4. 414 ■1 B : . ' 4 . B 3 ■ ’ 4. G 4. 7 4 0la B 4 • 2 1, . B ) /' 3. 9 4. 8 4. 39

L-, 3 B ■ i • 4 . 18 3 H 4. 3 •9 4 0

ly 4 ;. a . B 3 > /| 0 7 4. 39LB 4 a 1.4 9 40 3. 8 4, 9 4 1919 i 0 8; . i ' 41 3. 8 4. 9 4 19' 0 4 9 4 . ■ 4 ' 4. 7' 4. 9 4 3981 ■1 3 8.8 ■ i . aa 4 3 4 8 4. b 4 99. a ' 1 4 . ' /: . a 44 4 9 4. 7 4 6

A .i ti(i B - t\ u .1 La m o t o r men Jur omen ta3 Tiadea L 90 I. u u.-.cli otit

2 8 9

A P P E N D I X B •- iV;o .su r . ' i n o n l o f I 'PV . . M r t i c i * ; d L a m u t e r u .

EXTRAOi i‘RO.-.i 'JRi'j L ( o,;'. l.u iuu)u.-. 1‘ l> I'u [)a,pi IJ. oim - single case

P a r t i c l eN u m b e r

Measu siuiani t ( m;n '■ A e

n

P a r t i c l eN u-'iiüt; r

M e n s u r e m c n t (m m ) A B M e a n

1 4 . 2 '9 4 • b 4. 9 4 . 7 92 4 . 0 .'4 9. a 9, 4 . 4 9

5 4 . 0 2| , 1 ■ 22 4. 3) 9. 4 . 9 9

'■) /]. ( ) . . 8 !. : .. o 0 9. 9 J . 2 9

8 4 . / ' ' . 1.2; 2 1 4. 9 9. 0 4 . 9 9u 4. 2 ' . 0 2| . , ' 28 2, 2 9 . 9. 99

' { t . , . ; . ' . ’ ‘; 9. 99H ' . 0 ' . a ' . 0 3 0 9. 7; 9 . 0 9 . 2 9

9 4 . 8 ' ' . 0 3 1. 9. 3 9 . 2 d

10 4 . 8 ' . 02'

11 3 o ' 2 , 4

12 4 . 1 2. 1

1) -1. / M • ' ' 4 . 0

14 4 . 0 - i * ‘ ' 4 , 2 2

19 4 . 0 /!. ■ i . 02

i<; ■'! • - i •

17 4 . 7 4 . 2 4 . 8

i 8 4 . i 1 . 8 4 . 2 ,

19 4 . 2 4. 8 4 .

2 0 4 . 0 ' 4 . 2 2

2 1 4 . 2 4 . 8 2. n2

22 4 . 7 a . 0 . 1 . 8 ' '

A and H : t, W > ) i i amet e r' measun. . ' i r umts made

a. t pO^ t o ! ■ a ( 2 1 o t h e r .

290

PPÏÏNDIX 3 - Me •su 'neut m ’ ■!'/ ;>ar I i e 1 e d 1 am e te rc .X T R ACT F R O M C 4 27 ’ ( iu.1 t.M 11 eu n :; fihte-pap ill oia.n - single Ga

P a r t i c l e Men N u m b e r A

au re Uull Ll lO'tl )iMoaa

i'e.rt ic le ïuiiaber

Me a A

s û r e m e n t (mm B M e a n

i 1.2 ] * 1 e ' 4 . 8 9.0 4 . 9

/'I . 0 'I.. ' 4.1 24 4 . y 9 , 2 . 0

3 4 . ■ 4." 4 . 2 ' 2 2 4 . 0 4.9 4 . 7 9

4 4 . • i . 1 4 . - 2 u 5.1 4 . 2 4 . 8 9

9 4M' 2 .0 1MV'. 27 7). 1 4 . 2 4 .3) 9

I* 4 . 7 4. ' i . u u 28 ‘ ). 0 4 . 9 4.997 4. . 0 4 . I J ■•'9 4Mb 4 . 3 4 • 48 ■! . 7 ■ 1 . M 4 . 7 2 30 4 . ') 4 , 2 4. ‘ Mb9 4M. 1 * 41 9 . 0 4 .9 4.92

10 4 . 8 1 . 9 . 0 9 . 0 9 . 0

!- 1. . 4 1 . 1 ' ' 3 ') 21. 2 9 .2 9 4 . 79

12 2. 0 4 . 8 4 44 4,2 9, 2 4 . 7

13 4 . 8 :, 8, ■1 . 8 42 4 , 8 4.8 4 . 8

14 9,0 9 . 9' 9.2919 4,9 0 4 . 22: 44' 9.0 4.9 4 . 9 9

M» 4 . 8 2.2) ! . “ 48 /î . 8 4 . 8 / | . 8

1 7 l . e 2i 4'-' 4 0 0 / 1 . 9 4 . 8 2

18 9.0 i . 8 4. '2 40 9 , 0 4.8 4.91 , ) 9 . 0 ■i . 8 i . 9 41 4.8 4 . 9 4 . 8 92 0 2 .U 2 . i 4." 9 . 0 4Mb 4M/92' 1 / ] . 9 4. 8 4.2' 4 4 9.0 4.8 4.92 2 9 . 0 4 . 8 44 9.0 4 . 8 4.9

A and B - I, V I I ; liaiuete';- men s urement a madea t 9 0 ° l u u ' e ;h. u t i i e

291

A P P E N D I X B - Me an Mi; ,'nt oi 4 'Y irarlicLo d i a m e t e r s .

E X T R A C T F R O M y l-PV A 1 on t-inoi no; ii. b ropa;£)illoraa — H ingle case

Par t i d e J4 e au laMii 11 t \ ID.J] ) P a r 1; i c 1 e M e a s u r e m e n t (mm)Numb er A j : Ml ::j n N n m u e r A B M e a n

1 4 . ) ' 4 . 0 4.6 2.0 4, 6 4.64 .7 ■ 1 . 8 4. 7- 44 4 . 6 2.0 4.6

3 4 .2 a . U 4.24 •'3 4.3 4.9 4.74 4 » 7 4.1 '1.2 b 4.7 4.9 4 . 8

3 4 • <9 4 u 4 , 3',» '4 7 4.7 4.0 4. 356 4M» ■■'i. 3 ■ P8 4.0 3. 3 4.737 . 0 4. ‘ 4 .7-' 42 4.0 4.9 4.438 4 . 2 /[ . '» 4. ( to 4.9 4. 0 4.452 ' 1 . 4 . t, ■ i . tl 4.0 3.0 4.3!. 0 fi. 0 4 , 4 . V t' ' 4. 3 4.2 .4. (j11 4 • 1 4 . 4 . 2 4' ■; t 4. 2 4.3 . 41, ' . 0 a . I :4| 4 . 4 4 . 6 4 . 4

d 4 . 6 ' . 0 4,2'' ■'» 4 4 . 4 3.0 4 . 6

14 Î . 4| . 7' 4 . 7 3. 3 3.01 2 d * V . i . O' 37 4 . 3 3 • 4 4.616 . 0 ' >. > ' .1) 4 6 3 . 4.3 4.7317 4 . B ■I . A) ■ 1. 1' ' ■ 32 4. 3 3 . 0 4. 7518 /l. 'f 4 0 ' 4j , 8' 40 3 . 0 4.3 4.63

12 4. ' ' . 1 4 .64 41 4 . 6 4.9 4.8520 2.0 a. 0 . 0 44 2.1 3 . 0 5.15d 2 . 0 4 . 4 '1.27' '13 4.1 4.9 3.0pp 4.2 t . ‘4 4.4 44 4.3 4.9 4.7

A and P - tv; a 4 i nmol 0 meaou r e men 13 mad e

a t 2 0 ° t o u- ioh o t f u ;*

2 9 2

AP.b ENDIX b - M--a.su ;o..it:n>n'v G» f : i'Y pariimil. O dlame t e r o .E X T R A C T FROul C B i'V / ( on l-i noons i l n rc papi 11 orna - single case)

■îi]'ar tic] e N umb er A ü

, mu 1 Mail

A ;■ i r t i c 1 e K Lurnje r

Measurenient (mm) A 3 M e a n

1 4 . 2 4 . 2 ;. 1 23 4 . 3 2 . 2 4 . 7 2

p 4 . 4) ' M'> ■1.7 ' 24 4 . J 4MP 4 . 6

3 / | , 2 ‘ „ 2 2n 4. 8 2 , 0 4 . 9

4 4M ■ ' 2j . 0 ) 2o 2. ' ' i . 7 4 . 9 5

' + " ' * 1 2 . Ü 2 . 2 2 . 1

b 4 . d ' 1, ) . 0 2 8 2 . 2 4 . 8 2 . 0

' [ ■1.0 u. 0 4. ' ' 2 pi 4 . 8 4 .71 4 . 8 5

8 M , ‘ 4 . :M) .1. d 4 . 2 4 . 8 2

2 S . - ■'! • ' ■i. 4 31 2 . 0 2 , 3 2 . 1 2

10 4 . 2 4. b 2 . 1 4 . 2 2 . 0

11 ' i . ( ■i. 0 4 . 6 2 3) 2 . 2 4 . 0 4 . 9

10 4M >. 0 d . 72 34 2 . 0 4 . 2 4 . 7 5

13 4 • '' '1 ’ ' 34' 4 . 2 2 . 0 4 . 7 5

14 ;. 2 t . " 'i MV2 3o 4 . 8 2 . 2 2 0 0

12 4 . 2 1.4 2| . bp 3'3 4. 7 2 , 3 2 . 0

1 b n . 0 ■1 . e 4. '2 38 4MV 4 .2 4 . 4 2

17 C , ' i 32 4 . 0 4 . 2 4 . 2 5

16 4." n , 0 4 . 72 40 2 . 0 4 . 8 4 . 9

12 ' , . 0 4..1 ■1. 7 41 4 . 2 4 . 0 4 . 4 5

2 0 2 . 0 4; . 0 c.O 42 4 . 2 4 . 6 4 . 5 2

21 2 . 2 /I . " 4 . t» 43 4. 0 3 . 9 3.952 / 4 . 0 4 . ' ' 4 4 3 .2 4.2 4 , 0 5

A nnn : tvn. 4 Inmo to '■ iiiea j.in 'o men t s made a b

20° Lu t' ' i i (» Lh(' r.

293

Al'i'ENDIX B ■- Mo -, n: .■ -.aon I f'J'V p,. i' L i o l e diaaieters.E X T R A C T F R ü M CiE'V 2 ( on ' .-r.uoon s f i b rc p a p i l l o m a - single case

P a r t i c l e IJumb er

Mo iru ro;>nn : 'nm ) 2 t r t i c leA h j.Pniii N i m u r

Mea s û re m e n t (mm) A B M e a n

2

()

7

ü

9

10

Ll

17

18

/:. 0

1 .0

. 0

, I '

. 0;

. 0

. L

1 4

12

A an! P t a. t' 20° to each other

d i n m o Lee i n e ; ; s u r e ;n e n t s made

^ 4

AJO'EN D IX b - Me ,.sa inen l • ) f 2.2'Y r l i c i e d i nm e t e r s ,

EXTRACT FROM up ' dug d k l n f)a p L11 üfiia - s l .n g l e c a s e ]

P a r t i e l e Me.■•..ni .n.ii P.'i e t i c i e M e a s u r e m e n t (nuNum ber A r> M ;a u N u m b e r A B Mean

1 2 . 0 ■.'.' i ‘d 0 3 2 . 2' 2 . 9 2 . 7

d 4 . 0 d . ' /| ■’ 4 2. ' » 2 . 2 2 . 2 2

3 2 . d d . d f 2 2 2 . 2 2 . 3 2 . 4

/| ' ! , • ■' .0 .'d . 0 2 . 0 2 . 2

d 2. d h ■ '7 2. ’ 6 . 2. 7

d. ' ' ■ . • ’ ■■’ .'H 2. 2 2 . 0 ■). 7

Y ; ■: . ■( 1 ' .'2 . 6 2 . 0 2 . 8

6 ; 0 a, 0 2 . 2 2 ,4

■' . 0 • -, 1 2. i b . L 2 . 9

Id' . . a' . ' ' a , b . 1 2. k 2. 62

1 I ' .1 a d 2 , 2 6 . 0 2 . 7 2

1.' a . 0 i d 34 0 . a' 6 . 2 2 . 9 2

13 d • ' y d 4 3d 2. 3 b . 0 2 . 6 2

14 » . 0 ' ..:d '1 ■;o o. 0 4 . 8 2 . 4

13 ; . ‘ 2 ' a ■ ’ 2 37 2 . 0 . 2 2l u - .1 38 2 . 2 'd. 0 ■•.82

l Y ' . 3 ' ’ • 2 ' ( ' ) 32 ') „ 11 . 8 2 . 7

L H p . !■ 2'd '10 2 . 8 2 . 6 2. 8

12 2 . d 2 41 2 . 8 2 . 2 2 . 62

2 0 ) • ' j n . 0 2 '2 42 2 3' 4 . 9 2 . 0 21 J. 2 ■ n !' •') 4 ‘; 1>. 0 6 . 0 2 . 5

.12 2 , 0 d 2 '■} '"1 2 . 0 6 , 2 2 . 6

4 2 2. 3 2 . 4 2

4 2 2 . 8 2 . " 2 . 7

4 / 2 . 2 2 . 9 2 . 52

1 0 2 . 2 u . 0 2 , 8

4 2 2 , 0 2 . 8 2 . 4

2 0 2 . 8 2 . 9 2 . 8 2

k -a. ! i d : 'à ' i i a I i u ' aa ! ■ m a . . u r a i n e a t j made■1.1 2 0 ' Lu a n i l u ' a 1 1 f .

295

A P P E N D I X B Mea-saremoiiL of BPV p a r t i c l e d i a m e t e r s

C a l c u l ai, ion of t rue m a g n i f i c a t i o n

S t a n d a r d g r id h a s111 e; r e ■ f o j - u ne i, u a 1 k n o w n d i ; ; t a n c e between iLnenelec t ronmio rograpli nioan m e a s u r e d d i s t a n c e

SD

nu m b e r3 9 .7t r u e in n i f i c a t i o n

2 l6 0 l in e s /n im

0 . 00046 3 mm

3. 969cm

0 .0 6 1 2 cm 75

0 .0 0 0 4 6 3

8 2 , 760 X

C o n v e r s i o n of HP Y part j. c 1 c; m g a s 11 r- e ni e n t s

Actual, on o L c c t r o n m i o r o g r a p h m e a s u r e m e n t s were d i v ided by the c a i c u l a t o d true mag'nif icati o n , 82, 760 to give a.otual RPV particle d i a m e t e r s in riyt.a:j tabu 1 ;i tcd in t;i b lo 2 1 1 n thesi s text.

295

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£ 3 3

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509

APPENDIX E BOVINE PAPILLOMA VIRUS IN VITRO TRANSFORMATION ASSAY RESULTS.

iSSAY NUMBER OFTOMBER SAMPLE CELLS REPLICATES 100^0 TD50

1 65819 1) 11s 4 5 .1 6

2. 65819 2) 11s 4 5.24

5 65819 3) 11s 4 5 .1 6

k 65819 4) 11s 4 5 .5 4

L 65819 5) 11s 4 5.24b 65819 6) 11S 4 5 .5 1

7 65819 7) 11s 4 5 .5 4

8 65819 8) 11s U 4.24

9 65819 9) 11s 4 5 .4 5

10 65819 10) 11s 4 5 .5 5

11 65819 1) 12s 4 5 .4 7

12 65819 2) 12s 4 5 .5 5

13 65819 3) 128 4 5 .6 0

14 65819 4) 128 4 5 .6 8

15 65819 5) 128 4 5 .5 5

16 65819 6) 128 4 5 .4 7

17 65819 7) 128 4 5 .7 8

18 65819 8) 128 4 5 .5 5

19 65819 9) 12s 4 5 .6 8

20 65819 10) 128 4 5 .4 7

21 65819 1) 12c 4 4.02

22 65819 2) 12c 4 5 .6 0

25 65819 3) 120 4 5 .4 7

24 65819 4) 120 4 5 .5 5

25 65819 5) 120 4 3 .6 0

26 65819 6) 120 4 3 .8 5

27 65819 7) 12c 4 3 .6 0

28 65819 8) 120 h 3 .4 7

29 65819 9) 120 4 3 .5 5

50 65819 10) 120 4 3 .5 5

3 1 0

APPENDIX E cont

ASSAYNUMBER SAMPLE CELLS

NUMBER OF REPLICATES LOG

10 TD50

31 66702 (1) 12s 4 4 .1 5

3? 66702 ( 2 ) 12s 4 4.21

33 66702 (5) 12s 4 4 .1 5

34 66702 (4) 12s 4 4 .3 0

35 66702 (5) 12s 4 4.2336 66702 (6) 12s 4 4.1137 66702 (7) 12s h 4.27

38 66702 (8) 12s U 4 .1 7

39 66702 (9) 12s 4 4.3340 66702 ( 10) 12s 4 4 .3 0

41 66702 (1) 11s 4 4 .3 0

42 66702 (2) 11s 4 4.11

43 66702 (1) 11s 4 4.174 4 66702 (4) 11s 4 4 .2 3

45 66702 (5) 11s 4 4 .1 7

46 66702 (6) 11s 4 4 .2 3

47 66702 (7) 11s 4 4 .3 048 66702 (8) 11s 4 4 .1 5

49 66702 (9) 11s 4 4 .3 8

50 66702 (10) 11s 4 4 .3 3

51 66702 (1) 12c L 4.23

52 66702 (2) 12c 4 4 .17

53 66702 (3) 12c 4 4 .30

54 66702 (4) 12c 4 4.1155 66702 (5) 12c 4 4 .30

56 66702 (6) 12c 4 4 .27

57 66702 (7) 12c 4 4 .1758 66702 (8) 12c U 4.11

59 66702 (9) 12C 4 4 .1760 66702 (10) 12c 4 4.23

311

APPENDIX E cont

ASSAYNUMBER SAMPLE CELLS

NUMBER OF REPLICATES LOG^Q TD50

61 66702 (1) 11s 5 4,162 66702 (1) 115 5 4,165 66702 ( 1) 11s 5 3-964 66702 (1 ) 115 5 1,165 66702 ( . ) 11s 5 4.1

C6 65819 (1 ) 13 M 5 3 .3

67 65819 (2 ) 13M 5 3 .7

68 65819 (3 ) 13M 5 3-169 66702 (1 ) 13M 5 4,3

70 53833 2M 16 4 .5

71 53833 5M 12 4 ,3

72 66702 F 5M 4 6 .0

7.5 66702 PF 5M 5 4 .2

74 66702 PF 11s 5 4.175 Dog

Papilloma 11s 10 076 1! 12c 10 077 II 12s 10 078 1 r 2K 10 079 EGA 11s 10 080 12C 10 081 12s 10 082 2M 10 083 I 3M 10 084 RGB 11s 10 085 12c 10 086 12s 10 087 2M 10 088 13M 10 089 FR 11s 10 3 .1

90 12c 5 2 .7

12

APPENDIX E cont

ASSAY NUMBER 01NUMBER SAMPLE CELLS REPLICATES &0G TD50

91 12s 5 2 .7

92 2M 5 3 .1

93 13M 5 2 .9

94 FA 11s 10 095 FB 11s 10 096 FA 12s 10 097 120 10 098 FB 12s 10 099 12c 10 0

100 APV(20# CS) 11s 5 1-5101 APV(SS) 11s 5 2 .7

102 APV(Po8t 10) 11s 5 1*5103 FA 11SI 11s 5 3-1104 Hamster F. 11s 5 0105 Ï 13 11s 5 0106 66702 purified 11s 5 5 .8

107 DP purified 11S 5 0108 RPCPV 11s 5 2 .9

109 EGA purified 11 s 5 0110 66702 n,p« 11S 5 4.1111 FC 11s 5 0112 ROC 11s 5 3.1

113 FRC 11s 5 2 .7

1l4 EGA purified 11s 5 0

115 EGA crude 11s 5 0

116 FE purified 11s 5 4.1

117 66702 n *v . 12c 5 4 .7 5

118 68891 Men 12C 5 0

119 82 Mer 12c 5 0

120 68888 W16 12c 5 0

313

APPENDIX E cont

ASSAYNUMBER

121

122

12?

124125126

127128

SAMPLE

6889? Wl6

68882 W16

68891 WI6

METNMTNMÏNM':

68886 WI6

CELLS

12c

12c

12c

12c

12s

1 Lv

1?M

12c

NUMBER OFREPLICATES LOG Q TD50

2.90

3-700

0

0

4.5

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