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The generation of defective interfering rubella virus particles Bohn, Ehleen M. 1980

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THE GENERATION OF DEFECTIVE INTERFERING RUBELLA VIRUS PARTICLES B.Sc. (Honors, Microbiology) University of B r i t i s h Columbia, 1975 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF MICROBIOLOGY We accept th i s thesis as conforming to the required standard by EHLEEN M. BOHN i n THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 © Ehleen M. Bohn, 1980 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ft 11 S O 6 A b s t r a c t R u b e l l a v i r u s (RV) has been pro p o g a t e d i n murine (L-2) f i b r o b l a s t s and v i r a l t i t e r s d e t ermined u s i n g a m o d i f i e d h e m a d s o r p t i o n a s s a y . The t i t r a t i o n r e s u l t s suggested t h a t r u b e l l a v i r u s p o p u l a t i o n s may be comprised o f s t a n d a r d and d e f e c t i v e v i r i o n s . RV s t o c k s were examined f o r t h e i r a b i l i t y t o i n t e r f e r e w i t h s t a n d a r d (low i n t e r f e r e n c e ) r u b e l l a v i r u s . High i n t e r f e r e n c e RV s t o c k s c o u l d reduce s t a n d a r d RV i n f e c t i v i t y by g r e a t e r t h a n 99%. A new p u r i f i c a t i o n p r o c e d u r e has been deve l o p e d i n o r d e r t o examine t h e v i r i o n s produced by h i g h and low i n t e r f e r e n c e RV s t o c k s . R u b e l l a v i r a l p a r t i c l e s have been p u r i f i e d u s i n g a d i s c o n t i n u o u s r e n o g r a f i n g r a d i e n t f o l l o w e d by a c o n t i n u o u s r e n o g r a f i n g r a d i e n t . These g r a d i e n t s a l l o w e d s e p a r a t i o n o f i n t a c t p u r i f i e d v i r i o n s from c o n t a m i n a t i n g h o s t membranous m a t e r i a l . G r e a t e r t h a n 90% o f the t o t a l o r i g i n a l i n f e c t i v i t y was r e c o v e r e d as determined by the hemadsorption ass a y . E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n demonstrated the p r e s e n c e o f i n t a c t r u b e l l a v i r i o n s . V i r i o n s from h i g h and low i n t e r f e r e n c e RV s t o c k s have been p u r i f i e d , the RNA e x t r a c t e d from the p u r i f i e d v i r i o n s , 1*5 l a b e l e d w i t h I , e l e c t r o p h o r e s e d on 5% p o l y a c r y l a m i d e g e l s i i i and s u b j e c t e d t o a u t o r a d i o g r a p h y . V i r i o n s from low i n t e r f e r e n c e RV s t o c k s were c o n t a i n e d i n one band a t a . 3 d e n s i t y f> = 1.19 gm/cc . The two s i n g l e - s t r a n d e d RNA m o l e c u l e s t h a t c o u l d be e x t r a c t e d from these v i r i o n s have to . . m o l e c u l a r w e i g h t s o f 2.95 and 2.80 x 10 d a l t o n s . V i r i o n s p u r i f i e d from h i g h i n t e r f e r e n c e RV s t o c k s were c o n t a i n e d i n a t l e a s t t h r e e bands a t d e n s i t i e s f> = 1.19, 1.17 and 1.15 . 3 gm/cc . RNA m o l e c u l e s w i t h m o l e c u l a r w e i g h t s o f 2.95 and 2.80 x 10 d a l t o n s c o u l d be e x t r a c t e d from the v i r i o n s 3 b a n d i n g a t a d e n s i t y p= 1.19 gm/cc . V i r i o n s banding a t t h e 3 l i g h t e r d e n s i t i e s f>= 1.17 and 1.15 gm/cc c o n t a i n e d RNA fc m o l e c u l e s o f 1.25 and 1.05 x 10 d a l t o n s i n m o l e c u l a r w e i g h t r e s p e c t i v e l y . A f r e s h i s o l a t e o f RV has been s e r i a l l y passaged i n L-2 c e l l s i n o r d e r t o observe the s e q u e n t i a l g e n e r a t i o n o f d e f e c t i v e v i r a l p a r t i c l e s . The v i r i o n s from each passage have been p u r i f i e d , the RNA i s o l a t e d , l a b e l e d and a n a l y z e d by PAGE. The m o l e c u l a r w e i g h t s o f t h e RNA m o l e c u l e s i s o l a t e d i n d i c a t e d t h e p o s s i b l e c y c l i c g e n e r a t i o n o f d e f e c t i v e v i r a l p a r t i c l e s . TABLE OF CONTENTS i v Page A b s t r a c t i i L i s t o f F i g u r e s v i L i s t o f T a b l e s v i i L i s t o f A b b r e v i a t i o n s v i i i Acknowledgments i x I n t r o d u c t i o n 1 M a t e r i a l s and Methods I. C e l l s and Media 6 I I . V i r u s 6 I I I . Hemadsorption a s s a y 7 IV. P u r i f i c a t i o n o f r u b e l l a 8 V. ELISA 9 V I . E l e c t r o n m i c r o s c o p y 9 V I I . R a d i o l a b e l i n g o f R u b e l l a RNA 10 V I I I . I s o l a t i o n o f R u b e l l a RNA 10 IX. I o d i n a t i o n o f R u b e l l a RNA 11 X. PAGE o f R u b e l l a RNA 12 X I . C a l i b r a t i o n o f PAGE 12 . X I I . Containment 13 I . The hemadsorption a s s a y 14 I I . R u b e l l a i n t e r f e r e n c e assay 20 I I I . P u r i f i c a t i o n o f r u b e l l a v i r u s 20 IV. I s o l a t i o n and i o d i n a t i o n o f r u b e l l a RNA 32 V. I s o l a t i o n o f D.I. p a r t i c l e s from h i g h i n t e r f e r e n c e r u b e l l a s t o c k s . . 3 5 V I . G e n e r a t i o n o f D.I. p a r t i c l e s on s e q u e n t i a l p a s s a g i n g o f r u b e l l a f o l l o w i n g p r i m a r y i s o l a t i o n 42 D i s c u s s i o n 51 B i b l i o g r a p h y 58 v i LIST OF FIGURES F i g u r e T i t l e Page 1 Von Magnus e f f e c t 2 2 Q u a n t i t a t i o n o f r u b e l l a u s i n g hemadsorption 16 ass a y 3 T i t r a t i o n o f r u b e l l a u s i n g the hem a d s o r p t i o n 18 a s s a y 4 T i t r a t i o n o f low and h i g h i n t e r f e r e n c e 21 r u b e l l a s t o c k s 5 R u b e l l a p u r i f i c a t i o n p r o t o c o l 25 6 I s o p y c n i c g r a d i e n t a n a l y s i s o f r u b e l l a 26 7 ELISA and HAd a c t i v i t y o f r u b e l l a v i r u s 28 8 E l e c t r o n m i c r o g r a p h o f r u b e l l a v i r i o n s 30 9 Procedure f o r i s o l a t i o n and i o d i n a t i o n o f 33 R u b e l l a RNA \uS 10 G-25 sephadex column chromatography o f I - 34 r u b e l l a RNA 11 I s o p y c n i c g r a d i e n t a n a l y s i s o f low and h i g h 36 i n t e r f e r e n c e r u b e l l a s t o c k s MS 12 Autoradiograms o f I - r u b e l l a RNA 39 13 I s o p y c n i c g r a d i e n t a n a l y s i s o f passage 2 43 r u b e l l a 14 I s o p y c n i c g r a d i e n t a n a l y s i s o f passage 3 44 r u b e l l a 15 I s o p y c n i c g r a d i e n t a n a l y s i s o f passage 4 45 r u b e l l a 16 I s o p y c n i c g r a d i e n t a n a l y s i s o f passage 5 46 r u b e l l a 17 I s o p y c n i c g r a d i e n t a n a l y s i s o f passage 6 47 r u b e l l a 18 Major RNA s p e c i e s g e n e r a t e d on s e q u e n t i a l 49 p a s s a g i n g o f r u b e l l a v i i LIST OF TABLES F i g u r e T i t l e Page I R u b e l l a v i r u s i n t e r f e r e n c e 23 I I Recovery o f r u b e l l a d u r i n g p u r i f i c a t i o n 31 I I I M o l e c u l a r w e i g h t o f D.I. r u b e l l a RNA 41 IV RNA i s o l a t e d from s e q u e n t i a l l y 48 passaged R u b e l l a V i r u s v i i i A b b r e v i a t i o n s used i n t h i s t h e s i s a r e : RV: R u b e l l a v i r u s SV: S i n d b i s v i r u s SFV: S e m l i k i F o r e s t v i r u s L-2: Murine f i b r o b l a s t s MEM: Mi n i m a l e s s e n t i a l medium FCS: F e t a l c a l f serum TNE: 0.15m NaCl, 50mM T r i s - H C I and ImM EDTA, pH 7.8 TNM: 0.15m NaCl, 50mM T r i s - H C I , ImM MgCl , pH 7 .8 TCA: T r i c h l o r o a c e t i c a c i d ELISA: Enzyme l i n k e d immunoadsorbant a s s a y HAd : Hemadsorption M . 0 . i : M u l t i p l i c i t y o f i n f e c t i o n SDS: Sodium d o d e c y l s u l f a t e BSA: Bovine serum albumin PAGE: P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s HA: H e m a g g l u t i n a t i o n p r o t e i n BHK-21 Baby hamster f i b r o b l a s t s NDV: Newcastle d i s e a s e v i r u s 0/N: Over n i g h t D. I . D e f e c t i v e i n t e r f e r i n g S r b c . : Sheep r e d b l o o d c e l l s ^ PBS: Phosphate b u f f e r e d s a l i n e ACKNOWLEDGMENTS I w i s h t o thank Dr. D. van A l s t y n e f o r her su p p o r t and encouragement t h r o u g h o u t t h i s p r o j e c t . S p e c i a l thanks go t o Dr. G. Spiegelman f o r h i s a d v i c e and h e l p f u l s u g g e s t i o n s . I am i n d e b t e d t o Da v i d Pope f o r s u p p l y i n g t h e c e l l monolayers used i n t h i s work. 1 I n t r o d u c t i o n D e f e c t i v e i n t e r f e r i n g v i r a l p a r t i c l e s D e f e c t i v e i n t e r f e r i n g (D.I.) v i r a l p a r t i c l e s as d e f i n e d by Huang have t h e f o l l o w i n g p r o p e r t i e s : (12,14) (1) t h e y c o n t a i n a p a r t o f the s t a n d a r d v i r a l genome and as such t h e y can be c o n s i d e r e d v i r a l d e l e t i o n mutants. (2) t h e y c o n t a i n t h e same v i r a l s t r u c t u r a l p r o t e i n s as the s t a n d a r d v i r u s . (3) because t h e y a r e v i r a l d e l e t i o n mutants t h e y a r e unable t o r e p l i c a t e on t h e i r own b u t can r e p l i c a t e i n the presence o f s t a n d a r d v i r u s . (4) a l t h o u g h t h e y r e q u i r e s t a n d a r d v i r u s t o r e p l i c a t e , a t the same time t h e y i n h i b i t s t a n d a r d v i r i o n r e p l i c a t i o n and a r e t h e r e f o r e i n t e r f e r i n g . The e f f e c t s o f D.I. p a r t i c l e s were f i r s t observed on s e r i a l u n d i l u t e d p a s s a g i n g o f i n f l u e n z a v i r u s i n embryonated eggs.(45) A c y c l i c d e c r ease i n the r a t i o o f i n f e c t i v i t y t o h e m a g g l u t i n a t i o n was observed and t h i s o b s e r v a t i o n i s now known as t h e von Magnus phenomenon. ( F i g u r e 1) I t was f u r t h e r demonstrated t h a t a s e r i a l l y passaged i n f l u e n z a p r e p a r a t i o n o f low i n f e c t i v i t y c o u l d i n h i b i t the growth o f an i n f l u e n z a p r e p a r a t i o n o f h i g h i n f e c t i v i t y . The v i r a l p a r t i c l e s from low i n f e c t i v i t y s t o c k s c o n t a i n e d l e s s n u c l e o p r o t e i n p e r v i r i o n than v i r i o n s i s o l a t e d from s t a n d a r d p r e p a r a t i o n s w i t h h i g h i n f e c t i v i t y ( 2 3 ) . S i n c e t h i s i n i t i a l o b s e r v a t i o n o f D.I. p a r t i c l e 2 Undiluted passages F i g u r e 1 3 g e n e r a t i o n by i n f l u e n z a v i r u s the pr e s e n c e o f D.I. p a r t i c l e s has been demonstrated i n almost every a n i m a l v i r u s system s t u d i e d . In most cases i t i s d i f f i c u l t t o o b t a i n pure p r e p a r a t i o n s o f D.I. p a r t i c l e s uncontaminated by s t a n d a r d v i r i o n s . T h i s i s due p r i m a r i l y t o t h e s m a l l d i f f e r e n c e s i n th e v i r i o n d e n s i t i e s . Under t h e s e c i r c u m s t a n c e s not o n l y i s i t d i f f i c u l t t o demonstrate the i n a b i l i t y o f t h e d e f e c t i v e v i r i o n s t o r e p l i c a t e on t h e i r own b u t i t i s a l s o i m p o s s i b l e t o e s t a b l i s h u n e q u i v o c a l l y t h a t the i n t e r f e r e n c e c h a r a c t e r i s t i c s o f a g i v e n s t o c k are due t o the d e f e c t i v e v i r i o n s . However t h e lower i n f e c t i v i t y o f a d e f e c t i v e c o n t a i n i n g s t o c k as compared t o a s t o c k f r e e o f d e f e c t i v e s i s c o n s i d e r e d t o be c o n s i s t e n t w i t h the pr e s e n c e o f D.I. p a r t i c l e s . R u b e l l a v i r u s R u b e l l a v i r u s (RV) i s a member o f the f a m i l y T o g a v i r i d a e . RV i s n o n c y t o p a t h i c and r e p l i c a t e s i n the h o s t c e l l c y t o p l a s m . The v i r i o n s mature on budding t h r o u g h the c y t o p l a s m i c membrane and are t h e r e f o r e enveloped. D i f f i c u l t i e s have been c o n t i n u a l l y encountered i n attemp t s t o propogate and c h a r a c t e r i z e the v i r u s . A v a r i e t y o f p r o c e d u r e s have been r e p o r t e d f o r r u b e l l a p u r i f i c a t i o n from i n f e c t e d t i s s u e c u l t u r e media. These p r o c e d u r e s i n c l u d e (a) c o n c e n t r a t i o n o f t h e v i r u s u s i n g ammonium s u l f a t e 4 p r e c i p i t a t i o n (1) (b) d i a l y s i s a g a i n s t p o l y e t h y l e n e g l y c o l (PEG)(4) and (c) c e n t r i f u g a t i o n on a d i s c o n t i n u o u s s u c r o s e g r a d i e n t f o l l o w e d by s e d i m e n t a t i o n t h r o u g h a c o n t i n u o u s s u c r o s e g r a d i e n t ( 2 2 ) . A l t h o u g h t h e r e have been numerous r e p o r t s o f r u b e l l a p u r i f i c a t i o n t h e r e are no c o n s i s t e n t d a t a c o n c e r n i n g t h e r e c o v e r y o f v i r u s i n f e c t i v i t y , the p u r i t y o f t h e v i r u s p r e p a r a t i o n s o r t h e number o f s t r u c t u r a l p r o t e i n s p r e s e n t i n t h e i n t a c t v i r i o n . The problems encountered i n p u r i f y i n g the v i r u s are f u r t h e r e x a c e r b a t e d by the low and v a r i a b l e y i e l d s o f v i r u s . V a r y i n g d e n s i t i e s ( p = 1.16 - 1.23 gm/cc ) have been r e p o r t e d f o r r u b e l l a v i r i o n s (26, 34, 37, 4 1 ) . Perhaps t h e s e d i s c r e p a n c i e s c o u l d be accounted f o r by a mixed p o p u l a t i o n o f s t a n d a r d and d e f e c t i v e p a r t i c l e s . The r u b e l l a genome c o n s i s t s o f a s i n g l e - s t r a n d e d o i n f e c t i o u s RNA s e d i m e n t i n g a t 40S i n 0.1 M s a l i n e a t 20 C. The m o l e c u l a r w e i g h t based on t h e s e d a t a was c a l c u l a t e d t o be 3.0 x 10 d a l t o n s ( 1 1 ) . RNA i s o l a t e d from p a r t i a l l y p u r i f i e d v i r i o n s and f r a c t i o n a t e d by r a t e z o n a l c e n t r i f u g a t i o n i n s u c r o s e d e n s i t y g r a d i e n t s i n d i c a t e d the b u l k o f RNA sedimented as a s h a r p band o f 38S ( 3 5 ) . In a d d i t i o n , however, a 25S RNA'se s e n s i t i v e component was always d e t e c t e d . The a u t h o r s have suggested t h a t t h i s 25S RNA c o u l d r e p r e s e n t t h e genome o f i n c o m p l e t e d e f e c t i v e v i r u s p a r t i c l e s . There has been an a d d i t i o n a l r e p o r t which s u g g e s t s the e x i s t e n c e o f D.I. R u b e l l a p a r t i c l e s ( 2 7 ) . 5 S i n d b i s v i r u s and S e m l i k i F o r e s t v i r u s b o t h t o g a v i r u s e s , have been s t u d i e d i n g r e a t e r depth than R u b e l l a and have been found t o g e n e r a t e d e f e c t i v e s w i t h r e l a t i v e ease (39). A l t h o u g h t h e r e has been no d i r e c t r e p o r t o f the presence o f R u b e l l a D.I. p a r t i c l e s , i n f o r m a t i o n i n t h e l i t e r a t u r e i n d i c a t e s t h a t R u b e l l a s h o u l d be a b l e t o g e n e r a t e D.I. p a r t i c l e s . The o b j e c t i v e o f t h i s t h e s i s was t o determine i f r u b e l l a v i r u s c o u l d g e n e r a t e d e f e c t i v e - i n t e r f e r i n g v i r a l p a r t i c l e s . M a t e r i a l s and Methods C e l l s and Media Murine f i b r o b l a s t s (L-2) c e l l s were a k i n d g i f t from W F l i n t o f f (Department o f B a c t e r i o l o g y and Immunology, U n i v e r s i t y o f Western O n t a r i o ) . The c e l l s were r o u t i n e l y o p r o p o g a t e d as monolayers a t 37 C i n a humid atmosphere c o n t a i n i n g 5% CO^ w i t h M i n i m a l E s s e n t i a l Medium (MEM) supplemented w i t h 10% f e t a l c a l f serum (FCS; Grand I s l a n d B i o l o g i c a l Company, GIBCO), 100 jug/ml s t r e p t o m y c i n and 100 I.U./ml p e n i c i l l i n . V i r u s R u b e l l a v i r u s (RV), s t r a i n 2872R, was o b t a i n e d from G.D.M K e t t y l s ( B r i t i s h Columbia P u b l i c H e a l t h L a b o r a t o r i e s , Vancouver, B r i t i s h Columbia) as the f i r s t passage a f t e r p r i m a r y i s o l a t i o n . Stock v i r u s was r o u t i n e l y p r e p a r e d by i n o c u l a t i n g s e m i c o n f l u e n t monolayers o f L c e l l s w i t h RV a m u l t i p l i c i t y o f i n f e c t i o n (m.o.i.) o f 0.01. A f t e r o a d s o r p t i o n a t 34 C i n a humid atmosphere c o n t a i n i n g 5% CO^ f o r 1 1/2 h r , a d d i t i o n a l medium was added and the f l a s k was o i n c u b a t e d a t 34 C f o r 6 days, a t which t i m e the c u l t u r e o s u p e r n a t a n t was c o l l e c t e d and f r o z e n a t -80 C. 7 The h e m a d s o r p t i o n assay S e r i a l d o u b l i n g d i l u t i o n s o f RV s u s p e n s i o n s were used t o i n f e c t c o n f l u e n t monolayers o f L-2 c e l l s grown i n t i s s u e c u l t u r e chamber s l i d e s (Lab Tek P r o d u c t s , D i v i s i o n o f M i l e s L a b o r a t o r i e s , I n c . , N a p e r v i l l e , I l l i n o i s 60540). Two-chamber s l i d e s were used. Each chamber r e c e i v e d a 50 pl a l i q u o t o f th e a p p r o p r i a t e RV d i l u t i o n . V i r u s was a l l o w e d t o adsorb f o r 1 h r a t 34°C and 2.5 ml o f medium and 50 jdl o f a 20% s u s p e n s i o n o f h e p a r i n i z e d sheep e r y t h r o c y t e s i n A l s e v e r ' s s o l u t i o n were added d i r e c t l y t o each chamber. The s l i d e s o were t h e n i n c u b a t e d f o r 24 hours a t 34 C. The p l a s t i c chambers were removed and each s l i d e was washed g e n t l y by immersion i n pH 7.4 Dulbecco p h o s p h a t e - b u f f e r e d s a l i n e (1954) a t room tem p e r a t u r e and examined m i c r o s c o p i c a l l y f o r hemadsorbing c e l l s . U n i n f e c t e d c o n t r o l monolayers were t r e a t e d i n an i d e n t i c a l f a s h i o n . Those samples p r e t r e a t e d w i t h r u b e l l a s p e c i f i c a n t i s e r u m (H . I . t i t e r 1:128) were i n c u b a t e d w i t h an e q u a l volume o f a 1/32 d i l u t i o n o f t h e a n t i s e r u m . The samples were i n c u b a t e d o f o r 1 h r . a t 34 C p r i o r t o a d s o r p t i o n on t h e L-2 monolayers. These samples were t h e n t r e a t e d i n an i d e n t i c a l manner t o the i n f e c t e d m o n olayers. In m i x i n g experiments an e q u a l volume o f a h i g h i n t e r f e r e n c e s t o c k was mixed w i t h a low i n t e r f e r e n c e s t o c k . These samples were then t r e a t e d i n an i d e n t i c a l f a s h i o n t o the r u b e l l a samples. 8 P u r i f i c a t i o n o f r u b e l l a L c e l l monolayers were i n f e c t e d a t an m.o.i. o f 0.01 and o i n c u b a t e d a t 34 C f o r 6 days i n MEM c o n t a i n i n g FCS, 3 p e n i c i l l i n , s t r e p t o m y c i n and H - u r i d i n e as d e s c r i b e d . The c u l t u r e s u p e r n a t a n t s were c o l l e c t e d and c e n t r i f u g e d a t 3000 x o g f o r 20 m i n u t e s . A l l p r o c e d u r e s were c a r r i e d out a t 4 C u n l e s s o t h e r w i s e s t a t e d . The s u p e r n a t a n t o b t a i n e d was r e c e n t r i f u g e d a t 100,000 x g f o r 3 h o u r s and t h e r e s u l t i n g p e l l e t was resuspended i n 0.2 ml TNE b u f f e r (0.15M NaCl, 50 mM T r i s - H C l and 1 mM EDTA, pH 7.8). T h i s sample was l a y e r e d onto a 16 ml 25-45% ( w t / v o l ) d i s c o n t i n u o u s R e n o g r a f i n - 6 0 ( D i a t r i s o a t e Meglumine, Squibb) g r a d i e n t p r e p a r e d w i t h TNE b u f f e r and c e n t r i f u g e d i n an SW 27 r o t o r a t 55,000 x g f o r 2 h r . The s i n g l e , s h a r p band a t t h e i n t e r f a c e was c o l l e c t e d , p e l l e t e d as d e s c r i b e d p r e v i o u s l y , resuspended i n 0.5 ml TNM b u f f e r (0.15 M NaCl, 50 mM T r i s - H C l , ImM MgCl , pH 7.8) and l a y e r e d on a 12 ml 30-45% ( w t / v o l ) c o n t i n u o u s R e n o g r a f i n g r a d i e n t p r e p a r e d w i t h TNM b u f f e r . A f t e r c e n t r i f u g a t i o n a t 200,000 x g f o r 3 h r , 0.5 ml f r a c t i o n s were c o l l e c t e d . A 25 ^ul a l i q u o t from each f r a c t i o n was p r e c i p i t a t e d w i t h t r i c h l o r o a c e t i c a c i d (TCA). The p r e c i p i t a t e s were r e t a i n e d on g l a s s - f i b e r paper d i s c s (Reeve A n g e l , N . J . ) , washed t h r e e t i m e s w i t h c o l d , 5% TCA, f o l l o w e d by 95% e t h a n o l , d r i e d and c o u n t e d . A second a l i q u o t was removed from each f r a c t i o n f o r an e n z y m e - l i n k e d immunoadsorbent a s s a y ( E L I S A ) . A p p r o p r i a t e 9 f r a c t i o n s were th e n p o o l e d , d i l u t e d w i t h TNM b u f f e r and c e n t r i f u g e d a t 1 0 0 , 0 0 0 x g f o r 3 hr t o remove t h e R e n o g r a f i n . ELISA - En z y m e - l i n k e d Immunoadsorbent Assay The ELISA was performed a c c o r d i n g t o the p r o c e d u r e d e s c r i b e d by V o l l e r ( 4 4 ) . A 5 0 J J I sample was removed from each f r a c t i o n o b t a i n e d from the c o n t i n u o u s g r a d i e n t . I t was d i l u t e d 1 / 1 0 i n c o a t i n g b u f f e r . D u p l i c a t e 2 0 0 jal a l i q u o t s o f t h e f i l t e r e d sample were then adsorbed t o w e l l s o f a m i c r o t i t e r p l a t e (Cooke L a b o r a t o r y P r o d u c t s , Dynatech L a b o r a t o r i e s I n c . , 9 0 0 S l a t e r s Lane, A l e x a n d r i a , Va. 2 2 3 1 4 ) . A f t e r c o a t i n g , a p r e d e t e r m i n e d 1 / 1 6 d i l u t i o n o f human a n t i r u b e l l a a n t i s e r u m (HI t i t e r = 1 / 1 2 8 ) was added t o each w e l l . A n t i b o d y b i n d i n g was measured u s i n g a p r e v i o u s l y d e t e r m i n e d 1 / 2 0 0 0 d i l u t i o n o f r a b b i t antihuman IgG (Flow L a b o r a t o r i e s ) l i n k e d t o a l k a l i n e phosphatase. The A ^ ^ w a s det e r m i n e d a f t e r 3 0 min i n c u b a t i o n a t room t e m p e r a t u r e . R e n o g r a f i n had no e f f e c t on ELISA a c t i v i t y . E l e c t r o n M i c r o s c o p y on G r a d i e n t F r a c t i o n s G r a d i e n t f r a c t i o n s were n e g a t i v e l y s t a i n e d by m i x i n g t h e sample w i t h an e q u a l volume o f 3 % p h o s p h o t u n g s t i c a c i d , pH 7 . 0 . The samples were p l a c e d on Formvar-carbon c o a t e d g r i d s and excess f l u i d was withdrawn. The g r i d s were examined i n a P h i l i p s EM 301 e l e c t r o n m i c r o s c o p e . R a d i o l a b e l i n g o f R u b e l l a RNA V i r u s t o be l a b e l e d was grown as p r e v i o u s l y d e s c r i b e d . A f t e r a d s o r p t i o n f o r 1 1/2 h r . a t 34°C, MEM c o n t a i n i n g 10% FCS, 100 jug/ml s t r e p t o m y c i n , 100 I.U./ml p e n i c i l l i n and 3 3 / i C i / m l H - u r i d i n e (New England N u c l e a r , Boston, M a s s a c h u s e t t s , U.S.A.) was added. V i r u s was h a r v e s t e d 6 days p o s t i n f e c t i o n . The r a d i o l a b e l i n g a i d e d i n d e t e c t i o n o f r u b e l l a a f t e r i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n . However the low s p e c i f i c a c t i v i t y o f t h e H - l a b e l and the poor v i r u s y i e l d s made i t n e c e s s a r y t o i o d i n a t e the RNA p r i o r t o f u r t h e r a n a l y s i s . I s o l a t i o n o f R u b e l l a RNA The i s o l a t i o n o f v i r a l RNA has been d e s c r i b e d ( 3 5 ) . F o l l o w i n g i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a p p r o p r i a t e peak f r a c t i o n s were p o o l e d , d i l u t e d w i t h TNM b u f f e r and c e n t r i f u g e d a t 100,000 x g f o r 3 h o u r s . The RNA was r e l e a s e d from the p u r i f i e d v i r i o n s by t r e a t m e n t w i t h TNE b u f f e r c o n t a i n i n g 1% SDS a t room temperature. The r e l e a s e d v i r a l o RNA was d e p r o t e i n i z e d by p h e n o l t r e a t m e n t (22 C/10 m i n . ) . The aqueous phase was removed and t h e NaCl c o n c e n t r a t i o n a d j u s t e d t o 1.0M. The n u c l e i c a c i d was p r e c i p i t a t e d by 3 o , , volumes o f e t h a n o l a t -20 C/O.N. The p r e c i p i t a t e was 11 p e l l e t e d , d r i e d and was t h e n ready f o r i o d i n a t i o n . I o d i n a t i o n o f R u b e l l a RNA The P r e n s k y m o d i f i c a t i o n (32) o f t h e Cummerford r e a c t i o n (6) was used f o r the i o d i n a t i o n o f r u b e l l a RNA as f o l l o w s : To 5 p l RNA s o l u t i o n 5 yul H x0 2 ^/ul b u f f e r e d t h a l l i c i o n s o l u t i o n was added. 125 5 j u l Na i o d i d e (Amersham) 10 j a l a c i d i f i c a t i o n r e a g e n t . o The r e a c t i o n m i x t u r e was i n c u b a t e d i n a water b a t h a t 60 C/15 m i n u t e s . The pH m o n i t o r e d t o pH 4.7. A f t e r 15 minutes i n c u b a t i o n 0.5 ml TNE s o l u t i o n c o n t a i n i n g 0.001M Na^So 3was added and t h e RNA i n c u b a t e d an a d d i t i o n a l 20-30 minutes a t o 60 C. The sample was t h e n c o o l e d and l o d m a t e d RNA s e p a r a t e d from u n r e a c t e d I by G-25 sephadex column chromatography on a 25 cm x 1 cm column. The RNA was e l u t e d u s i n g ammonium a c e t a t e 0.1 M pH 5.0 b u f f e r c o n t a i n i n g 0.2% SDS. A l l r e a c t i o n m i x t u r e s were e i t h e r p r e t r e a t e d w i t h b e n t o n i t e (8) or c o n t a i n e d 0.2% SDS t o i n a c t i v a t e any c o n t a m i n a t i n g RNA'se. A l l g l a s s w a r e had been s t e r i l i z e d f o r 30 minutes and g l o v e s were worn d u r i n g manual m a n i p u l a t i o n s . E l u t e d 1 ml f r a c t i o n s were c o l l e c t e d f o l l o w i n g G-25 Sephadex column chromatography. A 10 j u l a l i q u o t was removed, 12 d i l u t e d 100 X and counted. F r a c t i o n s c o n t a i n i n g t h e i o d i n a t e d RNA were p o o l e d and the RNA p r e c i p i t a t e d by 3 volumes o f e t h a n o l a t -20 C/ON i n the pre s e n c e o f 100 L i g c o l d c a r r i e r y e a s t RNA. The d r i e d p r e c i p i t a t e was resuspended i n sample b u f f e r . A s m a l l a l i q u o t was removed t o de t e r m i n e RNA'se A s e n s i t i v i t y (Sigma C h e m i c a l s ) . A f t e r one hour i n c u b a t i o n a t 37°C i n the presence o f 10 / l g / m l RNA 1se A TCA p r e c i p i t a b l e counts were determined i n the presence o f c o l d c a r r i e r BSA. In a l l cases l e s s than 15% o f o r i g i n a l c ounts were p r e c i p t a b l e f o l l o w i n g RNA'se A d i g e s t i o n . PAGE - P o l y a c r y l a m i d e s l a b g e l e l e c t r o p h o r e s i s I - l a b e l e d RNA samples were resuspended xn TBE sample b u f f e r (45mM T r i s , 40mM b o r i c a c i d and 1.24mM EDTA). The samples were a p p l i e d t o a 10 cm. s l a b g e l ( 5 % a c r y l a m i d e and 6 M u r e a made up i n TBE). The RNA was e l e c t r o p h o r e s e d f o r 3 hours a t 25 ma. The g e l was then f i x e d f o r 1 h r . a t 22 C i n 25% i s o p r o p a n o l and 10% a c e t i c a c i d . The g e l was d r i e d onto f i l t e r paper b a c k i n g and s u b j e c t e d t o a u t o r a d i o g r a p h y u s i n g Kodak NS-2T f i l m . Exposure t i m e depended on the number o f counts a p p l i e d t o t h e g e l . The time would v a r y from 3-14 days exposure. C a l i b r a t i o n o f t h e PAGE system The a c r y l a m i d e g e l s were c a l i b r a t e d u s i n g t h r e e m o l e c u l a r w e i g h t m a r k e r s . RNA was e x t r a c t e d from S i n d b i s v i r u s (a k i n d g i f t from P i e r r e T a l b o t , Department o f B i o c h e m i s t r y , U.B.C.) i n an i d e n t i c a l manner t o r u b e l l a . In 6 6 M u r e a S i n d b i s RNA has a M.W. o f 2.1 x 10 d a l t o n s . E. c o l i fa ribosomes were p r e p a r e d and t h e 30S (1.0 x 10 d a l t o n s ) and 50S (1.67 x 10 d a l t o n s ) r i b o s o m a l RNA i s o l a t e d ( 1 8 ) . S i n d b i s RNA and E. c o l i rRNA were i o d i n a t e d as r u b e l l a RNA. These i o d i n a t e d markers were then e l e c t r o p h o r e s e d w i t h r u b e l l a RNA i n o r d e r t o c a l i b r a t e the g e l . Containment A l l m a n i p u l a t i o n s i n v o l v i n g r u b e l l a were conducted i n a c e r t i f i e d B l e v e l v i r a l containment hood. I o d i n a t i o n r e a c t i o n s were c a r r i e d out i n a fume hood p r o t e c t e d w i t h l e a d s h i e l d s . R e s u l t s I The h e m a d s o r p t i o n a s s a y R u b e l l a (RV) i s a n o n c y t o p a t h i c v i r u s and d i r e c t c y t o p a t h i c a s s a y s are not e f f e c t i v e i n t i t e r i n g the v i r u s . Q u a n t i t a t i o n has been t r a d i t i o n a l l y based on m i c r o f o c i f o r m a t i o n (7, 40) or t h e i n t e r f e r e n c e p r i n c i p l e (25, 2 8 ) . The i n t e r f e r e n c e a s s a y i s l e n g t h y and t e d i o u s . P r i o r i n f e c t i o n w i t h r u b e l l a r e n d e r s a c e l l immune t o s u p e r i n f e c t i o n w i t h N ewcastle D i s e a s e V i r u s (NDV). C e l l s u n i n f e c t e d w i t h r u b e l l a succumb t o the c y t o p a t h i c e f f e c t s o f NDV. The r u b e l l a i n f e c t e d c e l l s are a l l o w e d t o grow i n t o c o l o n i e s which are then enumerated t o o b t a i n r u b e l l a t i t e r s . The development o f a r a p i d and a c c u r a t e q u a n t i t a t i o n t e c h n i q u e became d e s i r e a b l e . The h e m a g g l u t i n a t i o n (HA) p r o t e i n i n r u b e l l a i n f e c t e d BHK-21 c e l l s has been r e p o r t e d (10, 3 8 ) . The e x p r e s s i o n o f t h e HA p r o t e i n on the c e l l s u r f a c e a l l o w s e r y t h r o c y t e s t o adsorb t o t h e s u r f a c e s o f r u b e l l a i n f e c t e d c e l l s . A h e m a d s o r p t i o n a s s a y has been developed which e n a b l e s n o n - c y t o p a t h i c mutants o f NDV t o be d e t e c t e d ( 2 4 ) . A m o d i f i c a t i o n o f t h i s a s s a y was d eveloped i n o r d e r t o d e t e c t n o n - c y t o p a t h i c w i l d t ype RV m i c r o f o c i formed i n r u b e l l a i n f e c t e d L-2 monolayers. The v i r u s i n f e c t e d monolayers were grown i n the p r e s ence o f sheep e r y t h r o c y t e s and 24 h o u r s p o s t i n f e c t i o n t h e hemadsorbing f o c i were enumerated. These hemadsorbing f o c i were easy t o s c o r e and t i t e r s o f i n f e c t i o u s r u b e l l a v i r u s c o u l d be o b t a i n e d ( F i g u r e 2 ) . A f t e r i n f e c t i o u s u n i t s were s c o r e d t h e y were p l o t t e d as a f u n c t i o n o f c o n c e n t r a t i o n and compared t o expected t h e o r e t i c a l r e s u l t s (2) ( F i g u r e 3 ) . The t h e o r e t i c a l e x p e c t ed r e s u l t s a r e e x p r e s s e d as e i t h e r o n e - h i t o r t w o - h i t k i n e t i c s . One-hit k i n e t i c s o c c u r s when the d e c r e a s e i n i n f e c t i o u s u n i t s i s p r o p o r t i o n a l t o the 1 s t power o f t h e v i r u s c o n c e n t r a t i o n . One v i r i o n i s a b l e t o i n i t i a t e an i n f e c t i o n . Two-hit k i n e t i c s r e s u l t s when the de c r e a s e i n i n f e c t i o u s u n i t s i s p r o p o r t i o n a l t o t h e 2nd power o f t h e c o n c e n t r a t i o n . Two p o p u l a t i o n s o f v i r i o n s a r e p r e s e n t and b o t h a re r e q u i r e d t o i n i t i a t e a s u c c e s s f u l i n f e c t i o n . A c o o p e r a t i v e e f f e c t i s o p e r a t i v e . When r u b e l l a i n f e c t i o u s u n i t s were p l o t t e d as a f u n c t i o n o f c o n c e n t r a t i o n the decrease i n HAd u n i t s was p r o p o r t i o n a l t o t h e f r a c t i o n a l power o f t h e c o n c e n t r a t i o n . These r e s u l t s s u ggested t h a t two p o p u l a t i o n s o f v i r i o n s may be p r e s e n t i n t h e v i r u s s t o c k and an i n t e r f e r e n c e e f f e c t might be o p e r a t i v e . V i r a l s t o c k s were then examined f o r t h e i r i n t e r f e r e n c e c h a r a c t e r i s t i c s . 16 F i g u r e 2A - Q u a n t i t a t i o n o f R u b e l l a v i r u s u s i n g the hemadsorption assay. C e l l s were i n f e c t e d w i t h s e r i a l d o u b l i n g d i l u t i o n s o f r u b e l l a and i n c u b a t e d i n t h e presence o f s r b c f o r 24 h o u r s a t 31 C. The hemadsorbing f o c i were v i s i b l e a f t e r removal o f t h e medium and t h r e e washes w i t h PBS. Arrows i n d i c a t e hemadsorbing f o c i . F i g u r e 2B - C o n t r o l c e l l s were i n c u b a t e d w i t h medium and t r e a t e d i n an i d e n t i c a l manner as the i n f e c t e d c e l l s . Only r e s i d u a l s i n g l e s r b c ' s remained a f t e r washing w i t h PBS. 17 18 F i g u r e 3. T i t r a t i o n o f r u b e l l a v i r u s u s i n g t h e hemadsorption assay. • • r u b e l l a HAd i n f e c t i o u s u n i t s p l o t t e d as a f u n c t i o n o f c o n c e n t r a t i o n . A A t h e o r e t i c a l e x p e c t ed r e s u l t s when the d e c l i n e i n t h e number o f i n f e c t i o u s u n i t s i s p r o p o r t i o n a l t o t h e f i r s t power o f the v i r u s c o n c e n t r a t i o n . (1 h i t k i n e t i c s ) . A A t h e o r e t i c a l e xpected r e s u l t s when the d e c l i n e i n the number o f i n f e c t i o u s u n i t s i s p r o p o r t i o n a l t o the second power o f the v i r u s c o n c e n t r a t i o n . (2 h i t k i n e t i c s ) . 19 \ v \ 1 1 1 1 f -8 16 32 64 Virus dilution F i g u r e 3 20 I I R u b e l l a i n t e r f e r e n c e assay V i r a l s t o c k s were t i t e r e d and i n f e c t i o u s u n i t s p l o t t e d as a f u n c t i o n o f c o n c e n t r a t i o n . Stocks which demonstrated h i g h degrees o f a u t o i n t e r f e r e n c e were mixed w i t h low a u t o i n t e r f e r e n c e s t o c k s . I n t e r f e r e n c e was measured as de c r e a s e i n i n f e c t i o u s u n i t s as a f u n c t i o n o f c o n c e n t r a t i o n . H ( F i g u r e 4 ) . When t h e h i g h i n t e r f e r e n c e s t o c k (4.48 x 10 HAd u n i t s / m l ) was mixed w i t h t h e low i n t e r f e r e n c e s t o c k (5.12 x 10 HAd u n i t s / m l ) i n f e c t i v i t y was reduced t o 1.60 x 10 HAd u n i t s / m l . P r e t r e a t m e n t o f a l l samples w i t h s p e c i f i c a n t i - r u b e l l a serum reduced the number o f i n f e c t i o u s c e n t e r s t o background l e v e l s . When the h i g h i n t e r f e r e n c e s t o c k was mixed w i t h the low i n t e r f e r e n c e s t o c k t h e r e was 99.7% i n h i b i t i o n o f v i r u s i n f e c t i v i t y ( T a b l e I ) . These r e s u l t s f u r t h e r suggested t h a t two p o p u l a t i o n s o f v i r i o n s , b o t h s t a n d a r d and d e f e c t i v e , might be p r e s e n t i n the h i g h i n t e r f e r e n c e s t o c k . V i r i o n s from b o t h h i g h and low i n t e r f e r e n c e s t o c k s were p u r i f i e d i n an attempt t o i s o l a t e t h e d e f e c t i v e p a r t i c l e s . I I I P u r i f i c a t i o n o f R u b e l l a v i r u s D i s c o n t i n u o u s and c o n t i n u o u s r e n o g r a f i n g r a d i e n t s were u t i l i z e d i n o r d e r t o s e p a r a t e i n t a c t v i r i o n s from c o n t a m i n a t i n g h o s t membranous m a t e r i a l . The v i r i o n s and the 21 F i g u r e 4. T i t r a t i o n o f low and h i g h i n t e r f e r e n c e r u b e l l a s t o c k s . • • T i t r a t i o n o f a h i g h i n t e r f e r e n c e RV s t o c k . 0 0 p r e t r e a t m e n t o f h i g h i n t e r f e r e n c e RV s t o c k w i t h s p e c i f i c a n t i - r u b e l l a serum ik- A T i t r a t i o n o f a low i n t e r f e r e n c e RV s t o c k . & & P r e t r e a t m e n t o f low i n t e r f e r e n c e RV s t o c k w i t h s p e c i f i c a n t i - r u b e l l a serum. • —• T i t r a t i o n o f low i n t e r f e r e n c e RV s t o c k mixed w i t h an e q u a l volume o f the h i g h i n t e r f e r e n c e RV s t o c k . O O P r e t r e a t m e n t o f t h e mixed low and h i g h i n t e r f e r e n c e s t o c k s w i t h s p e c i f i c a n t i - r u b e l l a serum. 22 100p 90J Virus dilution F i g u r e 4 TABLE I RUBELLA VIRUS INTERFERENCE VIRUS STOCK HAd UNITS/ml Low interference 5.12 x 10 H High interference 4.48 x 10 Low & high interference 1.6 x 10 2 4 h o s t c o n t a m i n a n t s have d i f f e r e n t d e n s i t i e s and t h e y c o u l d be e a s i l y s e p a r a t e d on t h e r e n o g r a f i n g r a d i e n t s . The p u r i f i c a t i o n p r o c e d u r e o u t l i n e d i n F i g u r e 5 was f o l l o w e d . F r a c t i o n s were c o l l e c t e d f o l l o w i n g i s o p y c n i c g r a d i e n t 3 c e n t r i f u g a t i o n . An a l i q u o t was removed t o measure H - u r i d m e i n c o r p o r a t i o n i n t o TCA p r e c i p i t a b l e counts ( F i g u r e 6 ) . A d d i t i o n a l a l i q u o t s d e t e r m i n e d ELISA a c t i v i t y ( F i g u r e 7A) and HAd i n f e c t i v i t y ( F i g u r e 7B). P u r i f i c a t i o n by i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n i n d i c a t e d 3 H - u r i d i n e i n c o r p o r a t i o n i n t o two peaks. ELISA a c t i v i t y d e t e c t e d r u b e l l a a n t i g e n s i n t h e s e two peaks. The HAd a s s a y demonstrated t h a t 90% o f o r i g i n a l v i r u s i n f e c t i v i t y was l o c a t e d i n t h e c e n t r e peak and l e s s than 2% was l o c a t e d i n the l i g h t d e n s i t y peak. E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n demonstrated th e p r e s e n c e o f i n t a c t v i r i o n s i n the c e n t r e peak. ( F i g u r e 8 ) . 3 S i n d b i s v i r u s which has a s i m i l a r d e n s i t y {(>= 1.196 gm/cc ) t o r u b e l l a v i r u s cosedimented w i t h t h i s peak. V i r u s r e c o v e r y was m o n i t o r e d a t each s t e p d u r i n g p u r i f i c a t i o n . G r e a t e r t h a n 90% o f o r i g i n a l i n f e c t i v i t y was r e c o v e r e d 1 X 5 ( T a b l e I I ) . P o l y a c r y l a m i d e g e l a n a l y s i s o f I - l a b e l e d v i r a l p r o t e i n s i n d i c a t e d t h a t h i g h m o l e c u l a r w e i g ht c o n t a m i n a n t s p r e s e n t i n s u c r o s e g r a d i e n t p u r i f i e d v i r i o n s were e l i m i n a t e d from v i r a l p r e p a r a t i o n s p u r i f i e d on r e n o g r a f i n g r a d i e n t s . The v i r u s p r e p a r a t i o n was c o n s i d e r e d pure as t h e r e was no f u r t h e r l o s s o f p o l y p e p t i d e s w i t h r e t e n t i o n o f 90% o f 25 V i r u s —y L-2 monolayer I H - u r i d i n e h a r v e s t 6 days p o s t i n f e c t i o n c o n c e n t r a t e by h i g h speed c e n t r i f u g a t i o n i . p e l l e t a p p l i e d t o d i s c o n i n u o u s R e n o g r a f i n g r a d i e n t band a t i n t e r f a c e p e l l e t e d i p e l l e t a p p l i e d t o c o n t i n u o u s R e n o g r a f i n g r a d i e n t 0.5 ml f r a c t i o n s c o l l e c t e d 2 5 i l l a l i q u o t p r e c i p i t a t e d w i t h 5% TCA 50 Jul f o r ELISA A c t i v i t y 5 0 7 * 1 * f o r HAd a s s a y . F i g u r e 5 26 F i g u r e 6 - I s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f R u b e l l a v i r u s . • • r u b e l l a i n f e c t e d p r e p a r a t i o n El- a mock i n f e c t e d p r e p a r a t i o n A A i o d i n a t e d S i n d b i s v i r u s Fraction number F i g u r e 6 28 F i g u r e 7A - ELISA a c t i v i t y o f r u b e l l a f o l l o w i n g i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n . • • r u b e l l a i n f e c t e d p r e p a r a t i o n D D mock i n f e c t e d p r e p a r a t i o n F i g u r e 7B - HAd a c t i v i t y o f r u b e l l a f o l l o w i n g i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n . • • r u b e l l a i n f e c t e d p r e p a r a t i o n 0 • mock i n f e c t e d p r e p a r a t i o n 7A Fraction number 30 F i g u r e 8A - E l e c t r o n m i c r o g r a p h o f r u b e l l a v i r i o n s c o n t a i n e d i n the c e n t r e peak f o l l o w i n g i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n . F i g u r e 8B - R u b e l l a c o r e s f o l l o w i n g T r i t o n X-100 t r e a t m e n t o f r u b e l l a v i r i o n s . 31 TABLE I I Recovery o f R u b e l l a d u r i n g p u r i f i c a t i o n VIRUS PREPARATION HAd U n i t s / m l T o t a l HAd % U n i t s Recovery 5 7 i n f e c t e d t i s s u e c u l t u r e 5.80 x 10 6.61 x 10 100 medium 7 7 sample a p p l i e d t o 3.96 x 10 6.34 x 10 95.8 d i s c o n t i n u o u s g r a d i e n t 7 7 band removed from 3.27 x 10 6.40 x 10 96.8 d i s c o n t i n u o u s g r a d i e n t 7 7 sample a p p l i e d t o 6.90 x 10 6.26 x 10 94.7 c o n t i n u o u s g r a d i e n t 5 & f r a c t i o n s 14-16 from 9.80 x 10 1.07 x 10 1.63 c o n t i n u o u s g r a d i e n t f r a c t i o n s 6-11 f r o c o n t i n u o u s g r a d i e n t 7 7 fr m 3.31 x 10 5.95 x 10 90.1 32 o r i g i n a l i n f e c t i v i t y . (43) These d a t a i n d i c a t e d t h a t the c e n t r e peak was comprised o f p u r i f i e d i n t a c t r u b e l l a v i r i o n s . Less t h a n 2% o f o r i g i n a l i n f e c t i v i t y was l o c a t e d i n t h e l i g h t d e n s i t y peak. I t was not c l e a r what t h i s peak was comprised o f . E l e c t r o n m i c r o s c o p i c e x a m i n a t i o n demonstrated t h e p r e s e n c e o f amorphous membranous m a t e r i a l . The i n f e c t i v i t y i n t h i s peak c o u l d be due t o i n c o m p l e t e v i r i o n s . R e s u l t s o f t h i s p u r i f i c a t i o n p rocedure i n d i c a t e d t h a t r e n o g r a f i n g r a d i e n t s would p e r m i t a h i g h degree o f p u r i f i c a t i o n w i t h a r e c o v e r y o f i n t a c t i n f e c t i o u s v i r i o n s up t o 90% o f t o t a l o r i g i n a l i n f e c t i v i t y . IV I s o l a t i o n and I o d i n a t i o n o f R u b e l l a RNA V i r i o n s from h i g h and low i n t e r f e r e n c e s t o c k s were p u r i f i e d and examined f o r the presence o f d e f e c t i v e p a r t i c l e s . The p r o t o c o l o u t l i n e d i n F i g u r e 9 was f o l l o w e d . In u n i n f e c t e d c o n t r o l samples, comparable f r a c t i o n s were p o o l e d t o peak f r a c t i o n s from i n f e c t e d p r e p a r a t i o n s . C o n t r o l and u n i n f e c t e d p r e p a r a t i o n s were t r e a t e d i n an i d e n t i c a l 1X5" manner. F o l l o w i n g i o d i n a t i o n i n f e c t e d and u n i n f e c t e d I-RNA m o l e c u l e s were e l u t e d o f f G-25 sephadex columns ( F i g u r e 1 0 ) . Column chromatography i n d i c a t e d t h a t '^I-RNA from i n f e c t e d samples e l u t e d o f f columns p r i o r t o the u n r e a c t e d i o d i n e . In u n i n f e c t e d samples o n l y u n r e a c t e d i o d i n e was e l u t e d o f f R u b e l l a v i r u s p u r i f i e d as d e s c r i b e d p r e v i o u s l y I peak f r a c t i o n s a r e p o o l e d and p e l l e t e d I RNA e x t r a c t e d by 1% SDS t r e a t m e n t I d e p r o t e i n i z a t i o n by phenol e x t r a c t i o n I (NaCl) a d j u s t e d t o 1.0 M I p r e c i p i t a t e RNA w i t h ETOH (-20 C 0/N) 125 l a b e l RNA w i t h I L s e p a r a t e r e a c t e d I-RNA from u n r e a c t e d I by G-25 sephadex column chromatography. p r e c i p i t a t e I-RNA w i t h ETOH (-20 C 0/N) p l u s c o l d c a r r i e r y e a s t RNA I W5 . . I-RNA p r e c i p i t a B u l k o f RNA r u n on 5% a l i q u o t t e s t e d f o r RNA'se a c r y l a m i d e g e l s (6M ur e a s e n s i t i v i t y and TBE b u f f e r system) ^ X TCA p r e c i p i t a b l e counts g e l s f i x e d , d r i e d d etermined s u b j e c t e d t o a u t o r a d i o g r a p h y F i g u r e 9 34 1 2 3 4 5 6 Fraction number F i g u r e 10. G-25LSephadex, column chromatography o f ' " i - r u b e l l a RNA -m e l u t i o n o f i n f e c t e d I - r u b e l l a RNA • — • e l u t i o n o f mock i n f e c t e d -RNA columns. A s m a l l a l i q u o t o f the p r e c i p i t a t e d v i r a l ' " i - R N A was removed and t e s t e d f o r RNA'se A s e n s i t i v i t y . In a l l i n s t a n c e s l e s s t h a n 15% o f the t o t a l counts remained f o l l o w i n g RNA'se A t r e a t m e n t . V. I s o l a t i o n o f D.I. P a r t i c l e s from h i g h i n t e r f e r e n c e  R u b e l l a s t o c k s H i g h i n t e r f e r e n c e and low i n t e r f e r e n c e R u b e l l a s t o c k s were o b t a i n e d . Both s t o c k s were p r e p a r e d by p a s s a g i n g a t a m.o.i. o f 0.01. The h i g h i n t e r f e r e n c e s t o c k was o b t a i n e d from passage 2 r u b e l l a and had a t i t e r o f 4.48 x 10 HAd u n i t s / m l . The low i n t e r f e r e n c e s t o c k was o b t a i n e d from if passage 6 r u b e l l a and had a t i t e r o f 5.14 x 10 HAd u n i t s / m l . The h i g h i n t e r f e r e n c e s t o c k was s e l e c t e d f o r i t s a b i l i t y t o reduce v i r a l i n f e c t i v i t y by 99.7%. The v i r i o n s from each s t o c k were p u r i f i e d and a n a l y z e d by i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n ( F i g u r e 11). The low i n t e r f e r e n c e s t o c k c o n t a i n e d one peak (A) which cosedimented w i t h S i n d b i s v i r u s a t a d e n s i t y ^= 1.19 gm/cc . The h i g h i n t e r f e r e n c e s t o c k c o n t a i n e d v i r i o n s banding a t t h r e e d e n s i t i e s . The most dense peak (A) cosedimented w i t h S i n d b i s v i r u s . The two l i g h t e r d e n s i t y peaks B and C had 3 d e n s i t i e s o f f> = 1.17 and 1.15 gm/cc r e s p e c t i v e l y . The v i r i o n s from each peak were p e l l e t e d , t h e RNA e x t r a t e d , l a b e l e d w i t h I , r u n on p o l y a c r y l a m i d e g e l s and s u b j e c t e d t o 36 Figure 1 1 A - Isopycnic gradient centrifugation of the low interference rubella stock. • • rubella infected preparation D— —• mock infected preparation Figure 1 1 B - Isopycnic gradient centrifugation of the high interference rubella stock. • • rubella infected preparation. Peaks A and B were pooled and recentrifuged to achieve better resolution. • • rub e l l a infected preparation. Peak C was pooled and recentrifuged to achieve better resolution. • 0 mock infected preparation indicates the position of cosedimented Sindbis virus marker. 3 7 Fraction number 38 a u t o r a d i o g r a p h y . ( F i g u r e 1 2 ) . The v i r i o n s i s o l a t e d from peak A o f the low i n t e r f e r e n c e 6 s t o c k c o n t a i n e d RNA m o l e c u l e s o f 2.95 and 2.80 x 10 d a l t o n s m o l e c u l a r w e i g h t . Repeated e x p e r i m e n t s c o n f i r m e d the p r e s e n c e o f t h e s e two RNA m o l e c u l e s i s o l a t e d from v i r i o n s b a n d i n g a t t h i s d e n s i t y . V i r i o n s i s o l a t e d from peak A o f the h i g h i n t e r f e r e n c e s t o c k c o n t a i n e d RNA m o l e c u l e s o f i d e n t i c a l m o l e c u l a r w e i g h t as t h o s e i s o l a t e d from peak A o f the low i n t e r f e r e n c e s t o c k s . V i r i o n s from the l i g h t e r d e n s i t y peaks b B and C c o n t a i n e d RNA m o l e c u l e s o f 1.25 and 1.05 x 10 d a l t o n s m o l e c u l a r w e i ght r e s p e c t i v e l y ( T a b l e I I I ) . These l i g h t e r d e n s i t y p a r t i c l e s l i k e l y r e p r e s e n t t r u e d e f e c t i v e p a r t i c l e s . The f o l l o w i n g d a t a would i n d i c a t e t h a t the d e f e c t i v e p a r t i c l e s were not a r t i f a c t s . T h e s e l i g h t e r d e n s i t y v i r i o n s were not p r e s e n t i n the low i n t e r f e r e n c e s t o c k . V i r i o n s o f o n l y one d e n s i t y were i s o l a t e d from the low i n t e r f e r e n c e s t o c k . The m o l e c u l a r w e i g h t s o f the RNA c o r r e l a t e d w e l l w i t h t h e d e n s i t y o f t h e p a r t i c l e s from which t h e y were i s o l a t e d . The s m a l l e r RNA m o l e c u l e s were not d e g r a d a t i o n p r o d u c t s . S i n g l e - s t r a n d e d S i n d b i s RNA t r e a t e d i n an i d e n t i c a l manner e l e c t r o p h o r e s e d t o a p o s i t i o n e q u i v a l e n t t o i t s t h e o r e t i c a l m o l e c u l a r w e i g h t . Most i m p o r t a n t , the 6 m o l e c u l a r w e i g h t o f the 2.95 x 10 d a l t o n RNA c o n t a i n e d i n 3 v i r i o n s b a n d i n g a t a d e n s i t y o f f = 1.19 gm/cc c o r r e l a t e d w e l l w i t h t h e 3.0 x 10 d a l t o n s r e p o r t e d i n the l i t e r a t u r e . Autoradiograms o f I-RNA i s o l a t e d from p u r i f i e d r u b e l l a v i r i o n s o f t h e : A. - low i n t e r f e r e n c e s t o c k . RNA i s o l a t e d from v i r i o n s banding a t Peak A. B. - h i g h i n t e r f e r e n c e s t o c k . RNA i s o l a t e d from v i r i o n s banding a t : Lane 1 - peak C Lane 2 - peak B Lane 3 - peak A 4 0 12 A 12 B 41 TABLE I I I M o l e c u l a r w e i g h t s o f D e f e c t i v e I n t e r f e r i n g R u b e l l a V i r u s R i b o n u c l e i c A c i d RNA Band Source (Peak) M o l e c u l a r Weight % o f T o t a l 6 ( F i g u r e 12) ( F i g u r e 11) x 10 d a l t o n s Genome 1 2 3 4 A A B C 2. 95 2.80 1.25 1.05 (100) 94 42 35 4 2 The d a t a demonstrated the p r e s ence o f l i g h t e r d e n s i t y d e f e c t i v e p a r t i c l e s i n h i g h i n t e r f e r e n c e r u b e l l a s t o c k s . These p a r t i c l e s were l i g h t e r i n d e n s i t y , c o n t a i n e d s m a l l e r m o l e c u l e s o f RNA whose s i z e c o r r e l a t e d t o the d e n s i t y o f t h e p a r t i c l e from which t h e y were i s o l a t e d , and t h e s e d e f e c t i v e p a r t i c l e s c o u l d o n l y be i s o l a t e d from s t o c k s e x h i b i t i n g h i g h i n t e r f e r e n c e c h a r a c t e r i s t i c s . V I The g e n e r a t i o n o f D.I, p a r t i c l e s on s e q u e n t i a l p a s s a g i n g  o f r u b e l l a f o l l o w i n g p r i m a r y i s o l a t i o n . The p r e s ence o f l i g h t e r d e n s i t y D.I. p a r t i c l e s was e s t a b l i s h e d i n a h i g h i n t e r f e r e n c e s t o c k . R u b e l l a was s e r i a l l y passaged t o examine the t r e n d o f d e f e c t i v e p a r t i c l e g e n e r a t i o n . More s p e c i f i c a l l y the a b i l i t y o f r u b e l l a v i r u s t o demonstrate a von Magnus e f f e c t was d e t e r m i n e d . A f r e s h l a b i s o l a t e was o b t a i n e d . R u b e l l a t i t e r s were always low and v a r i a b l e and a more s e n s i t i v e method was needed t o f o l l o w t h e g e n e r a t i o n o f D.I. p a r t i c l e s . The v i r i o n s from each passage were p u r i f i e d by i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n ( F i g u r e s 13 - 1 7 ) . The RNA was e x t r a c t e d , l a b e l e d w i t h I - i o d m e and a n a l y z e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( T a b l e I V ) . The major s p e c i e s o f RNA g e n e r a t e d a r e i n d i c a t e d i n F i g u r e 18. E x a m i n a t i o n o f t h e RNA m o l e c u l e s e x t r a c t e d from v i r i o n s a t each s u c c e s s i v e passage r e v e a l e d t h a t d e f e c t i v e p a r t i c l e s were r a p i d l y g e n e r a t e d . W i t h i n two passages o f i s o l a t i o n l 1 1 r 5 10 15 20 Top Fraction number F i g u r e 13 - i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f passage 2 r u b e l l a ^ p o s i t i o n o f s t a n d a r d r u b e l l a n • r u b e l l a i n f e c t e d • n mock i n f e c t e d V f r a c t i o n s i s o l a t e d f o r RNA e x t r a c t i o n 4 4 F i g u r e 14 - I s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f passage 3 r u b e l l a p o s i t i o n o f s t a n d a r d r u b e l l a r u b e l l a i n f e c t e d - p o o l e d / r e c e n t r i f u g e d • — • r u b e l l a i n f e c t e d - p o o l e d / r e c e n t r i f u g e d • • mock i n f e c t e d V f r a c t i o n s i s o l a t e d f o r RNA e x t r a c t i o n 45 6__ Fraction number F i g u r e 15 - I s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f p a s s a g e 4 r u b e l l a ^ p o s i t i o n o f s t a n d a r d r u b e l l a M • r u b e l l a i n f e c t e d • — • mock i n f e c t e d V f r a c t i o n s i s o l a t e d f o r RNA e x t r a c t i o n . 4 6 Fraction number F i g u r e 16 - I s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f passage 5 r u b e l l a b p o s i t i o n o f s t a n d a r d r u b e l l a • R r u b e l l a i n f e c t e d • • mock i n f e c t e d V f r a c t i o n s i s o l a t e d f o r RNA e x t r a c t i o n 4.7 6 I 4J E Q. O 3. I X CO 5 I 10 15 2 0 Top Fraction number F i g u r e 17 - I s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s o f passage 6 r u b e l l a • — • r u b e l l a i n f e c t e d 0 0 mock i n f e c t e d V f r a c t i o n s i s o l a t e d f o r RNA e x t r a c t i o n s TABLE IV R u b e l l a Passage F r a c t i o n ( s ) Peak Number I s o l a t e d Number M o l e c u l a r w e i ght o f RNA x 10" d a l t o n s S t a n d a r d R u b e l l a 2. 95 2.80 Passage 2 2.95 2.80 12 - 14 1.30 1.05 Passage 3 6 - 8 2.95 2. 80 9 - 1 1 1.25 1.05 12 - 15 1.05 Passage 4 2.40 1.60 1.00 0.80 2.95 2. 80 Passage 5 2.95 2.80 8 - 9 2. 30 1.50 12 - 13 not s u c c e s s f u l Passage 6 6 - 8 2.95 2.80 2 3 4 5 6 Passage number F i g u r e 18 - Major s p e c i e s o f RNA g e n e r a t e d on s e q u e n t i a l p a s s a g i n g o f r u b e l l a v i r u s . I n a l l passages a s m a l l amount o f s t a n d a r d RNA c o u l d be i s o l a t e d . • e x p e c t e d RNA m o l e c u l e s t o be i s o l a t e d from passage 1 p u r i f i e d v i r i o n s . s m a l l d e f e c t i v e m o l e c u l e s o f RNA were g e n e r a t e d . The f o l l o w i n g passages g e n e r a t e d v i r i o n s which had a d e n s i t y 3 g r e a t e r t h a n P= 1.19 gm/cc . A d i v e r s e range o f RNA m o l e c u l e s were i s o l a t e d from t h e s e dense v i r i o n s . R a p i d l y t h e r e a f t e r RNA m o l e c u l e s o f 2.95 and 2.80 x 10 d a l t o n s were r e g e n e r a t e d . These r e s u l t s i n d i c a t e d t h a t R u b e l l a conforms t o the a c c e p t e d d e f i n i t i o n o f D.I. p a r t i c l e g e n e r a t i o n . R u b e l l a demonstrated a r a p i d c y c l i c f l u c t u a t i o n i n y i e l d s o f s t a n d a r d and d e f e c t i v e v i r i o n s . These o b s e r v a t i o n s suggested t h a t r u b e l l a a l s o e x h i b i t s t h e von Magnus e f f e c t on c o n t i n u e d p a s s a g i n g o f t h e v i r u s . 51 D i s c u s s i o n R u b e l l a v i r u s has been t i t r a t e d u s i n g a m o d i f i e d h e m a d s o r p t i o n assay. T i t r a t i o n r e s u l t s suggested t h a t v i r u s s t o c k s c o u l d be comprised o f two p o p u l a t i o n s o f v i r i o n s , b o t h s t a n d a r d and d e f e c t i v e where an i n t e r f e r e n c e e f f e c t was o p e r a t i v e o r t h e r e c o u l d be v i r a l a g g r e g a t i o n a t lower d i l u t i o n s . The former i n t e r p r e t a t i o n was most l i k e l y as v i r a l i n f e c t i v i t y was reduced by g r e a t e r t h a n 99% when a h i g h i n t e r f e r e n c e s t o c k was mixed w i t h a low i n t e r f e r e n c e s t o c k . There i s a one hundred f o l d r e d u c t i o n i n v i r u s i n f e c t i v i t y when t h e h i g h i n t e r f e r e n c e s t o c k i s mixed w i t h t h e low i n t e r f e r e n c e s t o c k . T h i s magnitude o f i n h i b i t i o n has been observed i n s e v e r a l d e f e c t i v e v i r a l systems (15, 19) R u b e l l a has been h i g h l y p u r i f i e d u s i n g two r e n o g r a f i n g r a d i e n t s which a l l o w 90% r e c o v e r y o f i n f e c t i v i t y . The p u r i f i e d v i r u s was found t o cosediment w i t h S i n d b i s v i r u s a t 3 a d e n s i t y ofp= 1.19 gm/cc and e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n demonstrated the pr e s e n c e o f i n t a c t r u b e l l a v i r i o n s . The v i r i o n s were i s o l a t e d from a broad square peak wh i c h may r e p r e s e n t p o o r l y s e p a r a t e d s t a n d a r d and d e f e c t i v e v i r i o n s . V i r i o n s have been p u r i f i e d from the h i g h i n t e r f e r e n c e and t h e low i n t e r f e r e n c e s t o c k s , the RNA e x t r a c t e d and a n a l y z e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . The low i n t e r f e r e n c e s t o c k c o n t a i n e d v i r i o n s banding a t one d e n s i t y p = 1.19 gm/cc . These v i r i o n s c o n t a i n e d RNA 6> <o m o l e c u l e s w i t h m o l e c u l a r w e i g h t s o f 2.95 x 10 and 2.80 x 10 d a l t o n s . The h i g h i n t e r f e r e n c e s t o c k c o n t a i n e d v i r i o n s b a n d i n g a t a minimum o f t h r e e d e n s i t i e s . Once a g a i n v i r i o n s banded a t a d e n s i t y p = 1.19 gm/cc and c o n t a i n e d RNA 6 lc m o l e c u l e s o f 2.95 x 10 and 2.80 x 10 d a l t o n s . The two c l a s s e s o f l i g h t e r d e n s i t y v i r i o n s b anding a t d e n s i t i e s o f 3 3 p - 1.17 gm/cc and 1.15 gm/cc c o n t a i n e d RNA m o l e c u l e s o f <o 1.25 and 1.05 x 10 d a l t o n s r e s p e c t i v e l y . I t would be u n l i k e l y t h a t t h e s e l i g h t e r d e n s i t y v i r i o n s were a r t i f a c t s because m i n i m a l d e g r a d a t i o n o c c u r r e d d u r i n g p u r i f i c a t i o n . V i r i o n s banding a t o n l y one d e n s i t y f> = 1.19 gm/cc were c o n s i s t e n t l y i s o l a t e d from low i n t e r f e r e n c e s t o c k s . The l i g h t e r d e n s i t y v i r i o n s c o u l d o n l y be i s o l a t e d from s t o c k s e x h i b i t i n g h i g h i n t e r f e r e n c e c h a r a c t e r i s t i c s . The p r e s ence o f d e f e c t i v e v i r i o n s c orresponded w i t h the i n t e r f e r i n g c h a r a c t e r i s t i c s o f t h e h i g h i n t e r f e r e n c e s t o c k . The v i r i o n s banding a t a d e n s i t y f> = 1.19 gm/cc c o n t a i n e d RNA m o l e c u l e s o f 2.95 x 10 d a l t o n s i n m o l e c u l a r w e i g h t . T h i s m o l e c u l a r w e i ght corresponded f a v o u r a b l y t o t h e m o l e c u l a r w e i g h t o f 3.0 x 10 d a l t o n s r e p o r t e d i n the l i t e r a t u r e . The 2.80 x 10 d a l t o n RNA m o l e c u l e may r e p r e s e n t a l a r g e d e f e c t i v e m o l e c u l e because: (1) t h i s m o l e c u l e was r e p e a t e d l y i s o l a t e d from p u r i f i e d v i r i o n s o f b o t h h i g h and low i n t e r f e r e n c e s t o c k s ; (2) the s i n g l e - s t r a n d e d RNA o f S i n d b i s v i r u s was t r e a t e d i n an i d e n t i c a l manner and c o n s i s t e n t l y appeared as a s i n g l e band on p o l y a c r y l a m i d e 53 g e l s . D e g r a d a t i o n o f RNA d i d not appear t o be a problem; (3) An a l t e r n a t e c o n f o r m a t i o n o f t h e 2.95 x 10 d a l t o n m o l e c u l e would seem u n l i k e l y under t h e 6M u r e a r e d u c i n g c o n d i t i o n s o f t h e p o l y a c r y l a m i d e g e l s . The v i r i o n s c o n t a i n i n g e i t h e r o f t h e s e two RNA m o l e c u l e s s h o u l d have minor d i f f e r e n c e s i n t h e i r d e n s i t i e s . The s l i g h t d i f f e r e n c e i n t h e i r d e n s i t i e s might be enhanced on s h a l l o w e r r e n o g r a f i n g r a d i e n t s . I f the v i r i o n s c o u l d be s e p a r a t e d and p u r i f i e d , t h e RNA c o u l d be a n a l y z e d on p o l y a c r y l a m i d e g e l s . The RNA a n a l y s i s c o u l d e s t a b l i s h i f t h e 2.80 x 10 d a l t o n m o l e c u l e r e p r e s e n t e d a t r u e d e f e c t i v e genome. The l i g h t d e n s i t y v i r i o n s i s o l a t e d from the h i g h i n t e r f e r e n c e s t o c k c o n t a i n e d d e f e c t i v e genomes. The 1.25 and (o 1.05 x 10 d a l t o n m o l e c u l e r e p r e s e n t 42% and 35% o f the s t a n d a r d genome r e s p e c t i v e l y . The v i r i o n s from which t h e s e m o l e c u l e s were i s o l a t e d were not degraded v i r i o n s and t h e r e i s no r e a s o n t o b e l i e v e t h a t t h e s e m o l e c u l e s r e p r e s e n t b degraded 2.95 x 10 d a l t o n RNA. There was no d e g r a d a t i o n o f S i n d b i s RNA and i n a d d i t i o n the m o l e c u l a r weight o f the d e f e c t i v e RNA c o r r e s p o n d s t o the d e n s i t y o f the p a r t i c l e from w h i c h t h e y were i s o l a t e d . D u r i n g t h e c o u r s e o f t h i s i n v e s t i g a t i o n s e v e r a l o b s e r v a t i o n s were made. Standard r u b e l l a s t o c k s f r e e o f d e f e c t i v e s were never o b t a i n e d . Low i n t e r f e r e n c e s t o c k s c o n t a i n e d v i r i o n s o f one d e n s i t y ^ >= 1.19 gm/cc . Two 54 m o l e c u l e s o f RNA c o u l d be i s o l a t e d from th e s e v i r i o n s . H i g h i n t e r f e r e n c e s t o c k s c o n t a i n e d v i r i o n s o f the same d e n s i t y 3 f> = 1.19 gm/cc and i n a d d i t i o n v i r i o n s o f two l i g h t e r d e n s i t i e s . These v i r i o n s c o n t a i n e d s m a l l e r m o l e c u l e s o f RNA. Only r u b e l l a s t o c k s c o n t a i n i n g t h e s e d e f e c t i v e p a r t i c l e s c o u l d e l i c i t i n t e r f e r e n c e w i t h a low i n t e r f e r e n c e s t o c k . I d e a l l y , one would l i k e t o c o m p l e t e l y s e p a r a t e s t a n d a r d and d e f e c t i v e v i r i o n s p r i o r t o c o n d u c t i n g m i x i n g e x p e r i m e n t s . T h i s i s n o t y e t p o s s i b l e u s i n g the t e c h n i q u e s documented h e r e . A d d i t i o n a l o b s e r v a t i o n s were l e s s c l e a r . The i n t e r f e r e n c e c h a r a c t e r i s t i c s o f a s t o c k c o u l d change on p a s s a g i n g . In some i n s t a n c e s l e s s t h a n 10 HAd u n i t s / m l were d e t e c t a b l e . Y i e l d s o f s t a n d a r d v i r u s c o u l d not be i n c r e a s e d by p a s s a g i n g a t low m.o.i's. S i m i l a r l y d e f e c t i v e p a r t i c l e s c o u l d not be g e n e r a t e d a t w i l l by s i m p l y p a s s a g i n g a t h i g h m. o . i . ' s . The p r e s e n c e o f b o t h s t a n d a r d and d e f e c t i v e p a r t i c l e s i n r u b e l l a p o p u l a t i o n s c o u l d w e l l account f o r the c o n s i s t e n t l y poor y i e l d s o f v i r u s and f o r the i n c o n s i s t e n t r e p o r t s o f v i r i o n d e n s i t y i n t h e l i t e r a t u r e . T h i s may e x p l a i n why passage a t low m.o.i.'s d i d not s i g n i f i c a n t l y r a i s e v i r a l t i t e r s . Some r u b e l l a d e f e c t i v e p a r t i c l e s may always be p r e s e n t and i f so some degree o f a u t o i n t e r f e r e n c e would always be i n e f f e c t . 55 The p r e s e n c e o f d e f e c t i v e i n t e r f e r i n g r u b e l l a p a r t i c l e s has been demonstrated. The g e n e r a t i o n o f d e f e c t i v e p a r t i c l e s was examined on s e r i a l p a s s a g i n g o f t h e v i r u s a f t e r primary-i s o l a t i o n . The i s o p y c n i c g r a d i e n t c e n t r i f u g a t i o n p r o f i l e s i n d i c a t e t h a t t h e s p e c i f i c a c t i v i t y o f -> H - u r i d i n e i s too low t o d e t e c t t h e v i r u s . Even i n the absence o f a v i s i b l e peak o f H - u r i d i n e i n c o r p o r a t i o n s t a n d a r d m o l e c u l e s o f RNA w i t h a (o m o l e c u l a r w e i g h t 2.95 x 10 d a l t o n s c o u l d always be i s o l a t e d from th e f r a c t i o n s w i t h a d e n s i t y c o r r e s p o n d i n g t o f>= 1.19 gm/cc. The s m a l l e r RNA m o l e c u l e s o f 2.80 x 10 d a l t o n s c o u l d a l s o be i s o l a t e d from th e s e v i r i o n s . These r e s u l t s were not unexpected as a s m a l l amount o f s t a n d a r d v i r u s must always be p r e s e n t t o a l l o w r e p l i c a t i o n o f t h e d e f e c t i v e p a r t i c l e s . D i f f e r e n t t y p e s o f d e f e c t i v e p a r t i c l e s were g e n e r a t e d on s e r i a l p a s s a g i n g o f r u b e l l a . D e f e c t i v e p a r t i c l e s were g e n e r a t e d w h i c h were n o t o n l y l i g h t e r i n d e n s i t y b u t were more dense t h a n the s t a n d a r d v i r i o n . T h i s l a t t e r o b s e r v a t i o n has been r e p o r t e d f o r S i n d b i s v i r u s ( 3 6 ) . There have been a d d i t i o n a l r e p o r t s o f v i r i o n s o f i n c r e a s e d d e n s i t y c o n t a i n i n g more th a n one m o l e c u l e o f RNA. In some cases up t o 5 o r 6 RNA m o l e c u l e s i n m u l t i p l e copy form have been i s o l a t e d from t h e s e p a r t i c l e s ( 3 , 1 7 ) . When t h e RNA m o l e c u l e s from s e r i a l l y passaged v i r i o n s 5 6 were examined a c y c l i c "von Magnus" e f f e c t was obs e r v e d . T h i s o b s e r v a t i o n has not been p r e v i o u s l y r e p o r t e d f o r r u b e l l a . Most s t u d i e s o f r u b e l l a i n v o l v e d the use o f s t a n d a r d l a b s t r a i n s which have been r e p e a t e d l y passaged over s e v e r a l y e a r s . Use o f a p r i m a r y i s o l a t e has p e r m i t t e d the v i s u a l i z a t i o n o f the c y c l i c "von Magnus" e f f e c t . R u b e l l a appears t o g e n e r a t e d e f e c t i v e p a r t i c l e s r a p i d l y . One c h a r a c t e r i s t i c o f t h e von Magnus phenomenon i s t h e g r a d u a l d e c r e a s e i n the c y c l i c y i e l d s o f s t a n d a r d and d e f e c t i v e v i r i o n s . As r u b e l l a g e n e r a t e s d e f e c t i v e p a r t i c l e s r a p i d l y i t i s p r o b a b l e t h a t o v er s e v e r a l a d d i t i o n a l passages t h i s c y c l i c t r e n d would d e c l i n e and t h e von Magnus e f f e c t would g r a d u a l l y d i s a p p e a r . The h o s t c e l l i s known t o p l a y an i m p o r t a n t r o l e i n the g e n e r a t i o n o f d e f e c t i v e p a r t i c l e s (21, 29). The f a c t o r s w h i c h a l l o w one c e l l t y p e t o p e r m i t d e f e c t i v e p a r t i c l e g e n e r a t i o n and another t o p r o h i b i t t h e i r g e n e r a t i o n are p o o r l y u n d e r s t o o d . L i t t l e work has been done on the r o l e o f the h o s t c e l l i n the g e n e r a t i o n o f d e f e c t i v e i n t e r f e r i n g v i r a l p a r t i c l e s . S e v e r a l p r o p o s a l s have been put f o r w a r d t o e x p l a i n the g e n e r a t i o n o f D.I. p a r t i c l e s (13, 16, 20) and the means by whi c h t h e y i n t e r f e r (30, 3 1 ) . Recent a n a l y s i s o f S i n d b i s v i r u s and S e m l i k i F o r e s t V i r u s RNA i n d i c a t e t h e r e i s a p o l y A sequence a t t h e 3 - OH t e r m i n u s o f t h e i r genomes (4, 33, 4 6 ) . These o b s e r v a t i o n s have a i d e d i n d e t e r m i n i n g the o r i g i n s o f D.I. p a r t i c l e g e n e r a t i o n ( 9 ) . I f such a sequence c o u l d be l o c a t e d on t h e s t a n d a r d r u b e l l a genome d e f e c t i v e RV-RNA m o l e c u l e s c o u l d be screen e d f o r the pr e s e n c e o f t h i s sequence ( 5 ) . T h i s i n f o r m a t i o n may r e v e a l the mechanism by which d e f e c t i v e i n t e r f e r i n g r u b e l l a v i r a l p a r t i c l e s a re g e n e r a t e d and u l t i m a t e l y t h e means by wh i c h t h e y i n t e r f e r . I n summary, r u b e l l a v i r u s has been p u r i f i e d from low and h i g h i n t e r f e r e n c e s t o c k s . D e f e c t i v e v i r i o n s c o u l d be i s o l a t e d from h i g h i n t e r f e r e n c e s t o c k s which (1) were l i g h t e r i n d e n s i t y ; (2) c o n t a i n e d s m a l l e r m o l e c u l e s o f RNA whose s i z e c o r r e l a t e d w i t h the d e n s i t y o f t h e v i r i o n s from which t h e y were i s o l a t e d ; (3) corr e s p o n d e d t o the i n t e r f e r i n g p r o p e r t i e s a s s o c i a t e d w i t h t h a t s t o c k . V i r i o n s i s o l a t e d from low i n t e r f e r e n c e s t o c k s c o n t a i n e d RNA m o l e c u l e s w i t h m o l e c u l a r lo w e i g h t s o f 2.95 and 2.80 x 10 d a l t o n s . F i n a l l y , s e r i a l p a s s a g i n g o f a p r i m a r y i s o l a t e o f r u b e l l a and e x t r a c t i o n o f RNA from the p u r i f i e d v i r i o n s i n d i c a t e t h a t r u b e l l a may a l s o e x h i b i t a von Magnus e f f e c t . 58 BIBLIOGRAPHY 1. B a r d e l e t t i , G., K e s s l e r , N. and Aymard-Henry, M. (1975) Morphology, b i o c h e m i c a l a n a l y s i s and n e u r a m i n i d a s e a c t i v i t y o f r u b e l l a v i r u s . A r c h . V i r o l . 49: 175 - 186. 2. Boyeye, A., M e l n i c k , J.L. and Rapp, F. (1966) SV40 -a d e n o v i r u s " h y b r i d s " : p resence o f two genotypes and t h e r e q u i r e m e n t o f t h e i r complementation f o r v i r a l r e p l i c a t i o n . V i r o l . 2_8: 56 - 70. 3. B r u t o n , C.J. and Kennedy, S.I.T. (1976) D e f e c t i v e i n t e r f e r i n g p a r t i c l e s o f S e m l i k i F o r e s t V i r u s : S t r u c t u r a l d i f f e r e n c e s between s t a n d a r d and d e f e c t i v e - i n t e r f e r i n g p a r t i c l e s . J . gen. V i r o l . 31: 383 - 393. 4. B r u t o n , C.J. P o r t e r , A. and Kennedy, S.I.T. (1976) D e f e c t i v e i n t e r f e r i n g p a r t i c l e s o f S e m l i k i F o r e s t V i r u s : I n t r a c e l l u l a r e vents d u r i n g i n t e r f e r e n c e . J . gen. V i r o l . 31: 397 - 416. 5. C l e g g , J.C.S. and Kennedy, S.I.T. (1974) P o l y a d e n y l i c a c i d sequences i n v i r u s RNA s p e c i e s o f c e l l s i n f e c t e d w i t h S e m l i k i F o r e s t v i r u s . J . gen. V i r o l 22,: 331 - 345. 6. Commerford, S.L. (1971) I o d i n a t i o n o f n u c l e i c a c i d s i n v i t r o . B i o c h e m i s t r y 10: 1993 - 2000. 7. Dudgeon, J.A., B u t l e r , N.R. and P l o t k i n , S.A. (1964) ' F u r t h e r s e r o l o g i c a l s t u d i e s on the r u b e l l a syndrome. B r i t . Med. J . 2j_ 155 - 160. 8. F r a e n k e l - C o n r a t , H. and S i n g e r , B. (1961) E f f e c t s o f b e n t o n i t e on i n f e c t i v i t y and s t a b i l i t y o f TMV-RNA. V i r o l o g y 14: 54 - 65. 9. G u i l d , G.M. and S t o l l a r , V. (1977) D e f e c t i v e - i n t e r f e r i n g p a r t i c l e s o f S i n d b i s v i r u s : sequence r e l a t i o n s h i p s between SV RNA and i n t r a c e l l u l a r d e f e c t i v e v i r a l RNAs . V i r o l o g y 7_7: 178 -188. 10. Halonen, P.E., Ryan, and S t e w a r t , J.A. (1967) R u b e l l a 5 9 h e m a g g l u t i n i n p r e p a r e d w i t h a l k a l i n e e x t r a c t i o n o f v i r u s grown i n s u s p e n s i o n c u l t u r e o f BHK-2, c e l l s . P r o c . Soc. Exp. B i o l . (N.Y.) 125: 162-167. 11. H o v i , T. and V a h e r i , A. (1970) I n f e c t i v i t y and some p h y s i c o c h e m i c a l c h a r a c t e r i s t i c s o f R u b e l l a v i r u s r i b o n u c l e i c a c i d . V i r o l o g y 42: 1 - 8 . 12. Huang, A.S. (1973) D e f e c t i v e i n t e r f e r i n g v i r u s e s . Ann. Rev. M i c r o b i o l . 27: 101 - 107. 13. Huang, A.S. (1977) V i r a l p a t h o g e n e s i s and m o l e c u l a r b i o l o g y . B a c t e r i o l . Rev. 41: 811 - 821 14. Huang, A.S. and B a l t i m o r e , D. (1977) D e f e c t i v e i n t e r f e r i n g a n i m a l v i r u s e s . Comprehensive V i r o l o g y 10: R e g u l a t i o n and G e n e t i c s : V i r a l gene e x p r e s s i o n and i n t e g r a t i o n : Chapter 2: 73-116. Ed. H. F r a e n k e l - C o n r a t and R. Wagner. 15. Kascak, R.J. and Lyons, M.J. (1978) Bunyamwerma v i r u s I I The g e n e r a t i o n and n a t u r e f o d e f e c t i v e i n t e r f e r i n g p a r t i c l e s . V i r o l o g y 89 539 - 546. 16. Kennedy, S.I.T. (1976) Sequence r e l a t i o n s h i p s between the genome and the i n t r a c e l l u l a r RNA s p e c i e s o f s t a n d a r d and d e f e c t i v e - i n t e r f e r i n g S e m l i k i F o r e s t v i r u s . J . M o l . B i o l . 108: 491 - 511. 17. 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(1980) The p u r i f i c a t i o n o f R u b e l l a v i r u s (RV) and d e t e r m i n a t i o n o f i t s p o l y p e p t i d e c o m p o s t i o n . V i r o l o g y , a c c e p t e d f o r p u b l i c a t i o n , August 27, 1980. 44. V o l l e r , A., B i d w e l l , D. and B a r t l e t t , A. (1976) Manual o f C l i n i c a l Immunology, Chapter 69: 505 - 512. Ed. N.R. Rose and H. Friedman. American S o c i e t y f o r M i c r o b i o l o g y . 44. Von Magnus (1951) P r o p o g a t i o n o f the PR8 s t r a i n o f i n f l u e n z a A v i r u s i n c h i c k embryos. I I . The f o r m a t i o n o f " i n c o m p l e t e " v i r u s f o l l o w i n g i n o c u l a t i o n o f l a r g e doses o f seed v i r u s . A c t a . P a t h o l . M i c r o b i o l . Scand. 28: 278 - 293. 45. Wiess, B., Goran, D., Cancedda, R. and S c h l e s i n g e r , S. (1974) D e f e c t i v e - i n t e r f e r i n g passages o f S i n d b i s v i r u s . 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