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Purification and serology of apple chlorotic leaf spot virus isolated from Prunus glandulosa Thompson, Daniel Andrew 1990

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P U R I F I C A T I O N A N D S E R O L O G Y O F A P P L E C H L O R O T I C L E A F S P O T V I R U S I S O L A T E D F R O M PRUNUS GLANDULOSA B y D A N I E L A N D R E W T H O M P S O N B . S c , T h e U n i v e r s i t y o f V i c t o r i a , 1 9 7 9 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( D e p a r t m e n t o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l 1 9 9 0 © D a n i e l A n d r e w T h o m p s o n , 1 9 9 0 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. ^ , P l a n t S c i e n c e Department of The University of British Columbia Vancouver, Canada Date A p r i l 1 7 , 1990 DE-6 (2/88) A b s t r a c t Two i s o l a t e s (A and B) of apple c h l o r o t i c l e a f s p o t v i r u s (ACLSV) a s s o c i a t e d with l i n e p a t t e r n symptoms i n Prunus, glandulosa were i s o l a t e d i n Chenopodium quinoa and p u r i f i e d by a simple method u s i n g b e n t o n i t e c l a r i f i c a t i o n , p o l y e t h y l e n e g l y c o l p r e c i p i t a t i o n , and caesium sulphate i s o p y c n i c c e n t r i f u g a t i o n . Symptoms on herbaceous hosts were as p r e v i o u s l y r e p o r t e d f o r ACLSV. No symptoms were observed on a range of ACLSV woody host i n d i c a t o r s . I s o l a t e s A and B had a coat p r o t e i n MW of 26 and 24.5 k i l o d a l t o n s r e s p e c t i v e l y , ssRNA of MW 2.6 x 10 6 d a l t o n s , and dsRNA of MW 5.6 x 10 6, 4.9 x 10 6, and 4.5 x 10 6 d a l t o n s . The p a r t i c l e widths were 12 nm and lengths were 7 82 nm (A) and 7 32 nm (B). Buoyant d e n s i t y i n caesium sulphate was 1.27 g/cc . In t h i n s e c t i o n s of C. quinoa, flexuous rods were seen i n the cytoplasm and nucleus of young s i e v e - t u b e members. P o l y c l o n a l a n t i s e r a prepared a g a i n s t the A and B i s o l a t e s had h i g h background r e a c t i o n s and r e q u i r e d c r o s s - a d s o r p t i o n with host sap. Three monoclonal a n t i b o d i e s (MAB) a g a i n s t i s o l a t e A d e t e c t e d Prunus s t r a i n s w h ile a f o u r t h d e t e c t e d both Malus and Prunus s t r a i n s i n C. quinoa with low background r e a c t i o n s . The broad spectrum MAB c o u l d not be used as a t r a p p i n g antibody but c o u l d be d i r e c t l y conjugated with a l k a l i n e phosphatase or used i n an i n d i r e c t t r i p l e - a n t i b o d y sandwich ELISA. Tobacco mosaic v i r u s (TMV) recovered from P. glandulosa was i d e n t i c a l i n s e r o l o g i c a l and p h y s i c a l p r o p e r t i e s t o the TMV-U1 type s t r a i n . i i i TABLE OF CONTENTS PAGE T i t l e Page i Abs t r a c t i i Table of Contents i i i L i s t of Tables i v L i s t of Figures v Acknowledgement v i CHAPTER 1. General I n t r o d u c t i o n 1 2. B i o l o g i c a l Detection . . . . 4 In t r o d u c t i o n 4 M a t e r i a l s and Methods 5 Res u l t s 8 Dis c u s s i o n 13 3. V i r u s P u r i f i c a t i o n 16 In t r o d u c t i o n 16 M a t e r i a l s and Methods 18 Resul t s 22 Dis c u s s i o n 28 4. Cytopathology and P h y s i c a l P r o p e r t i e s 32 In t r o d u c t i o n 32 M a t e r i a l s and Methods 33 Resul t s 37 Dis c u s s i o n 46 5. S e r o l o g i c a l Detection 50 In t r o d u c t i o n 50 M a t e r i a l s and Methods 51 Resul t s 55 Dis c u s s i o n 63 6. I s o l a t i o n of Tobacco Mosaic V i r u s . . . . 66 In t r o d u c t i o n 66 M a t e r i a l s and Methods 66 Results 67 Disc u s s i o n 70 7. General D i s c u s s i o n 71 References C i t e d 75 i v LIST OF TABLES TABLE PAGE 1. The herbaceous host range of ACLSV i s o l a t e s A and B. 9 2. The e f f e c t s of buffer additives on apple c h l o r o t i c leafspot virus l o c a l lesion number i n Chenopodium quinoa3. 12 3. Physical c h a r a c t e r i s t i c s of viruses from P. glandulosa 44 4. Comparison of infected/healthy absorbance r a t i o i n double antibody sandwich ELISA for cross-adsorbed versus non cross-adsorbed apple c h l o r o t i c leafspot virus antiserum 56 5. Comparison of three grinding buffers and three methods of d i l u t i n g conjugate on the absorbance r a t i o of infected/healthy C.quinoa i n double antibody sandwich ELISA 59 6. Reactions of four monoclonal antibodies (MAB) to seven i s o l a t e s of apple c h l o r o t i c leafspot virus i n Chenopodium quinoa by t r i p l e antibody sandwich ELISA 60 7. The ef f e c t s of six d i f f e r e n t grinding buffer additives on the detection of apple c h l o r o t i c leafspot virus i s o l a t e A i n Chenopodium quinoa and Prunus glandulosa by t r i p l e antibody sandwich ELISA 62 The herbaceous host range of tobacco mosaic virus (TMV) i s o l a t e d from Prunus glandulosa 69 V LIST OF FIGURES FIGURE PAGE 1. Symptoms induced by apple c h l o r o t i c l e a f s p o t v i r u s ( i s o l a t e B) i n Prunus glandulosa (A) and Chenopodium quinoa (B) 10 2. P u r i f i e d i s o l a t e B ACLSV from a contaminated p r e p a r a t i o n showing spheres. Bar = 100 nm 24 3. SDS-PAGE of cros s - a d s o r p t i o n (C/A) of i s o l a t e A.. . 28 4. Modal length d i s t r i b u t i o n of 46 p a r t i c l e s of i s o l a t e A ACLSV measured from p u r i f i e d p r e parations 38 5. Modal length d i s t r i b u t i o n of 38 p a r t i c l e s of i s o l a t e B ACLSV measured from p u r i f i e d p r e parations 39 •6. V i r u s - l i k e p a r t i c l e s i n apple c h l o r o t i c l e a f s p o t v i r u s i n f e c t e d t i s s u e s 41 7. E l e c t r o p h o r e t i c v i s u a l i z a t i o n of dsRNA e x t r a c t e d from apple c h l o r o t i c l e a f s p o t i n f e c t e d Chenopodium quinoa. . 42 8. E l e c t r o p h o r e t i c v i s u a l i z a t i o n of ssRNA e x t r a c t e d from apple c h l o r o t i c l e a f s p o t i n f e c t e d Chenopodium quinoa 43 9. E l e c t r o p h o r e t i c v i s u a l i z a t i o n and Western b l o t of apple c h l o r o t i c l e a f s p o t v i r u s coat p r o t e i n . . . . 45 10. Agar g e l double d i f f u s i o n t e s t with p u r i f i e d apple c h l o r o t i c l e a f s p o t v i r u s i s o l a t e s A and B.. . 57 v i ACKNOWLEDGEMENTS Many people deserve thanks f o r h e l p i n g i n the completion of t h i s t h e s i s . Thanks to the Food Production and Insp e c t i o n Branch of A g r i c u l t u r e Canada f o r a l l o w i n g me to persue t h i s degree and to the A g r i c u l t u r e Canada, Vancouver Research S t a t i o n s t a f f f o r the use of t h e i r f a c i l i t i e s and equipment, and f o r t h e i r advice and i n f o r m a t i o n . Thanks to the members of my committee, Dr. Copeman, Dr. Hansen and Dr. Hudson f o r t h e i r h e l p f u l comments and guidance. Thanks t o my research advisor Dr. Stace-Smith who taught me many t h i n g s , but above a l l showed me what one can accomplish w i t h d e d i c a t i o n and hard work. F i n a l l y , t h i s t h e s i s i s dedicated t o my wife Barbara and daughters Lauren and J u l i a . I thank them f o r t h e i r support and s a c r i f i c e s t h a t have allowed me to complete t h i s work and I hope tha t someday I can repay them. Chapter 1 - General I n t r o d u c t i o n 1 V i r u s d i s e a s e s of t r e e f r u i t s were r e c o g n i z e d as e a r l y as the e i g h t e e n t h c entury. In France, sour c h e r r y yellows was d e s c r i b e d i n 1768 and peach yellows was d i s c o v e r e d on a P h i l a d e l p h i a farm i n 1791 (60). In t h i s century much has been l e a r n e d about the e t i o l o g y , e p i p h y t o l o g y , and p h y s i c a l c h a r a c t e r i s t i c s of t r e e f r u i t v i r u s e s . Some d i s e a s e s p r e v i o u s l y a t t r i b u t e d t o v i r u s e s were found t o be caused by mycoplasmas, r i c k e t t s i a or v i r o i d s . The v i r u s e s r e s p o n s i b l e f o r many d i s e a s e s were i s o l a t e d , p u r i f i e d , and t h e i r p h y s i c a l and s e r o l o g i c a l p r o p e r t i e s e s t a b l i s h e d . In s p i t e of these advances the e t i o l o g i c a l agents f o r many v i r u s - l i k e d i s e a s e s of t r e e f r u i t remain unknown and the d i s e a s e s are d e t e c t e d only by t h e i r g r a f t - t r a n s m i s s i o n to s u s c e p t i b l e i n d i c a t o r s . In c o n t r a s t to stone f r u i t v i r u s e s , many pome f r u i t l a t e n t v i r u s e s cause no symptoms i n the common f r u i t i n g v a r i e t i e s . Examples are apple c h l o r o t i c l e a f s p o t (ACLSV), apple stem g r o o v i n g and apple stem p i t t i n g v i r u s e s . These v i r u s e s may reduce growth and v i g o u r depending on v a r i e t y and r o o t s t o c k (8). Although some evidence e x i s t s f o r n a t u r a l spread of these v i r u s e s (65,71) they are spread mainly by g r a f t t r a n s m i s s i o n and t h e i r importance i s due to t h e i r widespread d i s t r i b u t i o n i n c l o n a l l y propagated stock. ACLSV occurs i n more t r e e f r u i t s p e c i e s than any other v i r u s and causes a wide range of d i s e a s e s r anging from symptomless 2 i n f e c t i o n s t o severe s t o c k / s c i o n i n c o m p a t i b i l i t y and f r u i t symptoms. In apple i t i s considered l a t e n t i n most f r u i t i n g c u l t i v a r s , however, some crabapples are s e n s i t i v e and are used as v i r u s i n d i c a t o r s (54,59). In Japan the c l o n a l l y propagated Maruba-kaido (Malus p r u n i f o l i a Borkh. var rinkii Rend.) rootstock i s widely used. ACLSV i n f e c t e d c u l t i v a r s on t h i s r o o t s t o c k show a general t r e e d e c l i n e with xylem p i t t i n g and phloem n e c r o s i s i n the r o o t s t o c k (58). Two diseases causing f r u i t symptoms i n apple, r i n g s p o t and russet r i n g have been a s s o c i a t e d w i t h ACLSV, the former i n combination w i t h apple stem p i t t i n g (60, 75) . ACLSV causes pear r i n g p a t t e r n mosaic (14). Symptoms c o n s i s t of l i g h t green r i n g s and l i n e p a tterns and, i n s e n s i t i v e c u l t i v a r s , the f r u i t shows sunken greenish r i n g s . I n c o m p a t i b i l i t y between i n f e c t e d pear c u l t i v a r s and quince r o o t s t o c k s , p a r t i c u l a r l y quince E, can occur (76). The diseases caused by ACLSV i n stone f r u i t s are more severe than i n pome f r u i t s and are more prevalent i n Europe than North America. Peach dark green sunken mottle v i r u s produces sunken spots and wavy l i n e s on peach leaves (12) and can cause s c i o n / stock i n c o m p a t i b i l i t i e s i n some c u l t i v a r s (53). Plum bark s p l i t v i r u s causes sunken dark areas on the stems which e v e n t u a l l y crack and s p l i t . The t r e e s become stunted w i t h a p r o l i f e r a t i o n of suckers and are uneconomical (21). Some ACLSV i n f e c t e d plum c u l t i v a r s show c h l o r o t i c r i n g s and l i n e s known as pseudopox symptoms (17). In a p r i c o t s ACLSV causes i n c o m p a t i b i l i t y with some s e e d l i n g r o o t s t o c k s (55) and pseudopox, where the f r u i t becomes 3 deformed wi t h a sunken dark n e c r o s i s (64). ACLSV i s l a t e n t i n most cherry c u l t i v a r s (30) but some are s e n s i t i v e and sunken n e c r o t i c spots develop on the f r u i t s (17). There i s one report of the v i r u s i n f e c t i n g Prunus glandulosa Thunb. ' s i n e n s i s ' and causing p i n p o i n t c h l o r o t i c l e s i o n s and n e c r o t i c l e a f t i p s (11). Other n a t u r a l host p l a n t s of ACLSV are Amelanchier canadensis Med., Chaenomeles spp., Crataegus monogyna Jacq., C. oxyacantha L. emend Jacq. and Mespilus germanica L. (74) . C o n t r o l of ACLSV i s based on the use of healthy propagative m a t e r i a l , both s c i o n and r o o t s t o c k . The virus, i s detected on herbaceous and woody host i n d i c a t o r s and by s e r o l o g i c a l techniques, however, one method may not be e f f e c t i v e f o r screening the v a r i o u s b i o l o g i c a l and s e r o l o g i c a l s t r a i n s . ACLSV i s r e l a t i v e l y easy to e l i m i n a t e by conventional hot a i r heat therapy (79) and can be e l i m i n a t e d by f o l i a r a p p l i c a t i o n of the a n t i - v i r a l drug r i b a v i r i n (36) or by i n c o r p o r a t i o n of the drug i n shoot c u l t u r e media (37). The purpose of t h i s study was to i d e n t i f y the sap-t r a n s m i s s i b l e v i r u s i s o l a t e d from dwarf f l o w e r i n g almond (P. glandulosa ' s i n e n s i s ' ) , to develop a s u i t a b l e s e r o l o g i c a l d e t e c t i o n technique, and to i n v e s t i g a t e the b i o l o g i c a l and s e r o l o g i c a l p r o p e r t i e s of t h i s v i r u s . P r e l i m i n a r y evidence from host range s t u d i e s and p a r t i c l e morphology suggested the v i r u s was a c l o s t e r o v i r u s , probably apple c h l o r o t i c l e a f s p o t v i r u s . 4 Chapter 2 - B i o l o g i c a l Detection I n t r o d u c t i o n Apple c h l o r o t i c l e a f s p o t (ACLSV) can be detected on both herbaceous and woody i n d i c a t o r p l a n t s . The v i r u s was f i r s t detected from apple with the i n d i c a t o r s Malus platycarpa Rehd. (54) and the Russian rootstock clone R12740-7A (58). In Prunus the recommended i n d i c a t o r i s GF 305 seedlings (Prunus persica (L.) Batsch) but others such as Prunus tomentosa Thunb. and Prunus domestica L. 'P 707' have been used. Pear r i n g p a t t e r n mosaic i s detected on quince C7/1 (Cydonia oblonga M i l l e r ) , or Pyrus communis L. 'Hardy' and 'Nouveau Po i t e a u ' . Many authors have p o i n t e d out the d i f f i c u l t i e s i n d e t e c t i n g the v a r i o u s ACLSV s t r a i n s w i t h one i n d i c a t o r . Marenaud et a l . (56) i n o c u l a t e d 9 ACLSV s t r a i n s from Malus, Prunus and Pyrus t o 11 woody i n d i c a t o r s and found, w i t h one exception, t h a t the Prunus s t r a i n s were not detected on R12740-7A. Only M. platycarpa detected a l l s t r a i n s while P. tomentosa and quince C7/1 detected a l l but one s t r a i n . S l i g h t l y d i f f e r e n t r e s u l t s were obtained by Paunovic (63) where M. platycarpa detected only 4 of 21 i s o l a t e s whereas peach GF 305 detected a l l i s o l a t e s . Dunez et a l . (21) reported t h a t the r e a c t i o n s of the i s o l a t e s from Malus and Prunus were g e n e r a l l y d i f f e r e n t but a few exceptions were found i n each genus wi t h some apple i s o l a t e s showing symptoms on Prunus i n d i c a t o r s and v i c e versa. Detection on woody i n d i c a t o r s i s f u r t h e r complicated by the uneven d i s t r i b u t i o n of the v i r u s i n the host. Generally, buds 5 near the proximal end of the shoot are more l i k e l y t o be i n f e c t e d while the t i p buds are often healthy (26). ACLSV i s sap t r a n s m i s s i b l e t o herbaceous p l a n t s (13). Chenopodium quinoa W i l l d . i s the most r e l i a b l e i n d i c a t o r p l a n t but other species such as Chenopodium amaranticolor Coste & Reyn. and Chenopodium murale L. are a l s o i n f e c t e d . Some ACLSV i s o l a t e s d e s c r i b e d as " s u p e r - l a t e n t " by Gilmer and Mink (31) are detected by C. quinoa but not the standard apple i n d i c a t o r s . The time of year and t i s s u e type i n f l u e n c e the r e l i a b i l i t y of ACLSV herbaceous t e s t i n g as r e s u l t s are v a r i a b l e l a t e r i n the growing season. Blossoms and young leaves are the best inoculum source (67,51). In C. quinoa, i n f e c t i v i t y i s decreased by an i n h i b i t o r y substance, probably a ribonuclease, which i s i n a c t i v a t e d by bentonite or the c o n c e n t r a t i o n reduced by d i l u t i o n of the i n f e c t e d sap (68). Just as r e s u l t s are v a r i a b l e on woody i n d i c a t o r s w i t h ACLSV i s o l a t e s from d i f f e r e n t species, the same i s t r u e on herbaceous hosts. Chairez and L i s t e r (9) and Paunovic (63) both found d i f f e r e n c e s i n symptom type and s e v e r i t y on C. quinoa w i t h i s o l a t e s from Malus and Prunus. M a t e r i a l s and Methods V i r u s I s o l a t e s . Two sources of dwarf f l o w e r i n g almond {Prunus glandulosa Thunb. ' s i n e n s i s ' ) were obtained from the A g r i c u l t u r e Canada, Pl a n t Quarantine S t a t i o n , Sidney, B r i t i s h Columbia. Accession Q1114-01 o r i g i n a t e d from Ontario, Canada and accession Q1292-01 6 was from Washington State, U.S.A. These are r e f e r r e d to as i s o l a t e A and i s o l a t e B r e s p e c t i v e l y . Both sources were maintained as pot t e d p l a n t s i n a glasshouse at 20-25 C during experimentation. Other i s o l a t e s of ACLSV used f o r c h a r a c t e r i z a t i o n of the monoclonal a n t i b o d i e s were propagated i n C. quinoa and i n c l u d e d the f o l l o w i n g : American Type Culture C o l l e c t i o n (ATCC) PV 60 from Malus (51); P 205 i s o l a t e d from Malus i n Japan (81); SI i s o l a t e d from Prunus avium L. by A. J . Hansen, Summerland, B.C.; UBC i s o l a t e d from Malus sylvestris M i l l . 'Spartan' at Vancouver, B.C.; and Q1000-04 i s o l a t e d from M. sylvestris ' K a t j a ' which o r i g i n a t e d i n Sweden. Transmission A f t e r i n i t i a l l o c a l l e s i o n t e s t s on Chenopodium quinoa to determine the optimum b u f f e r f o r rub i n o c u l a t i o n , i n f e c t e d t i s s u e was r o u t i n e l y ground i n 0.01 M T r i s , pH 8.5, wi t h 0.01 M MgS04 (TM b u f f e r ) and rubbed on t e s t p l a n t s p r e v i o u s l y dusted with carborundum. For i s o l a t i o n s from woody host leaves, flowers and r o o t s , 1.0% n i c o t i n e was added to the b u f f e r and the p l a n t s were r i n s e d w i t h water a f t e r being i n o c u l a t e d . Both A and B i s o l a t e s were passed twice through s i n g l e l o c a l l e s i o n s t o reduce the chance of a mixed i n f e c t i o n . Host range s t u d i e s were done using i n f e c t e d C. quinoa sap and p u r i f i e d v i r u s . A l l t e s t p l a n t s were back i n o c u l a t e d to C. quinoa and assayed by ELISA t o detect symptomless i n f e c t i o n s . The f o l l o w i n g herbaceous p l a n t s were t e s t e d w i t h the A and B i s o l a t e s : Beta vulgaris L. ' D e t r o i t Dark 7 R e d ' , Chenopodium amaranticolor C o s t e & R e y n . , C. murale L . , C . quinoa W i l l d . , Cucumis sativa L . ' S t r a i g h t 8 ' , Datura stramonium L . , Gomphrena globosa L . , Lactuca sativa L . , Nicotiana benthamniana D o m i n . , Nicotiana clevlandii G r a y , N. glutinosa L . , Nicotiana occidentalis W h e e l e r ( a c c e s s i o n u n k n o w n ) , Nicotiana tabacum L . ' H a r r o w n o v a ' , ' X a n t h i ' , ' S a m s u n ' , Nicotiana rustica L . , Petunia hybrida H o r t . V i l m . ' C o r a l S a t i n ' , Phaseolus vulgaris L . ' B l a c k T u r t l e ' , Vigna unguiculata ( L . ) W a l p . , a n d Zinnia elegans J a c q . T h e e f f e c t s o f t h e pH o f t h e g r i n d i n g b u f f e r on t r a n s m i s s i o n was i n v e s t i g a t e d u s i n g TM b u f f e r a t pH 6 . 0 , 6 . 5 , 7 . 0 , 7 . 5 , 8 . 0 , a n d 8 . 5 a n d i n o c u l a t i n g C. quinoa i m m e d i a t e l y a n d 24 h r l a t e r . To t e s t t h e e f f e c t s o f d i l u t i o n o n symptom e x p r e s s i o n i n C. quinoa, i s o l a t e A i n f e c t e d t i s s u e was g r o u n d 1 : 3 (w/v) i n TM b u f f e r a n d s e r i a l l y d i l u t e d i n f i v e t w o - f o l d s t e p s t o 1 : 9 6 . T h e s i x s a m p l e s w e r e i n o c u l a t e d t o C. quinoa. To d e t e r m i n e symptoms o n woody h o s t i n d i c a t o r s , b u d s f r o m e a c h i s o l a t e w e r e T - b u d d e d o n t o f i e l d i n d i c a t o r s (two p e r t e s t ) . Pome f r u i t i n d i c a t o r s h a d b o t h t h e i n d i c a t o r a n d i n o c u l u m b u d s s i m u l t a n e o u s l y a p p l i e d t o t h e r o o t s t o c k w h i l e Prunus i n d i c a t o r b u d s w e r e a p p l i e d t h e p r e v i o u s summer . A l s o , some p o t t e d ' r o o t s t o c k s w e r e d o u b l e b u d d e d w i t h t h e i n d i c a t o r a n d t h e v i r u s i n f e c t e d s o u r c e i n A u g u s t , moved t o 4 C i n O c t o b e r a n d b r o u g h t i n t o a 20 C g l a s s h o u s e i n J a n u a r y f o r symptom e x p r e s s i o n . T h e A a n d B i s o l a t e s w e r e b u d d e d t o t h e f o l l o w i n g woody i n d i c a t o r s : Prunus avium ' B i n g ' , ' S a m ' ( f i e l d ) ; P . serrulata L i n d l e y 8 'Kwanzan' ( f i e l d ) ; P. persica 'Halford' ( f i e l d ) ; P. persica ' S i b e r i a n C seedlings (greenhouse); P. armeniaca L. ' T i l t o n ' ( f i e l d ) ; Pyronia veitchii (Trab.) Guillaum (greenhouse); Malus platycarpa (greenhouse); M. sylvestris 'Spy 227', 'R12740-7A' (greenhouse) . R e s u l t s The host ranges f o r the A and B i s o l a t e s were narrow, i n f e c t i n g only three of twenty species t e s t e d (Table 1). No cases were found of symptomless i n f e c t i o n s . The A and B i s o l a t e s produced l o c a l l e s i o n s on C. quinoa a f t e r 2 weeks and systemic symptoms a f t e r 2-4 weeks ( F i g . I B ) . I f p l a n t s were i n o c u l a t e d at a young stage (four l e a v e s ) , systemic symptoms appeared sooner than when o l d e r p l a n t s were i n o c u l a t e d . In C. amaranticolor the B i s o l a t e produced only systemic symptoms and no l o c a l l e s i o n s whereas the A i s o l a t e showed only l o c a l l e s i o n s . Back i n o c u l a t i o n of symptomless C. amaranticolor leaves to C. quinoa gave negative r e s u l t s . The A i s o l a t e was unique i n t h i s respect as the f i v e other i s o l a t e s a l s o became systemic i n C. amaranticolor. Transmissions from P. glandulosa t o C. quinoa could be made with leaves, flowers or r o o t s . Both P. glandulosa mother p l a n t s showed a c h l o r o t i c l i n e p a t t e r n and v e i n break symptom which increased i n i n t e n s i t y as the season progressed ( F i g . 1 A). Since TMV was recovered from only one of these sources, i t i s assumed th a t the symptoms were not a s s o c i a t e d w i t h TMV but could be a t t r i b u t e d to ACLSV. Without T a b l e 1. T h e h e r b a c e o u s h o s t r a n g e o f i s o l a t e s A a n d B . 9 Symptoms p r o d u c e d b y i s o l a t e S p e c i e s A B Beta vulgaris 0 0 Chenopodium amaranticolor n e c 11 s y s l e s d i s t , l p C. murale c h l 11 s y s l e s , s y s l e s d i s t C. quinoa n e c 1 1 n e c 11 s y s l e s s y s l e s mo, l p mo, l p Cucumis sativa 0 0 Datura stramonium 0 0 Gomphrena globosa 0 0 Lactuca sativa 0 0 Nicotiana benthamniana 0 0 N. clevlandii 0 0 N. glutinosa 0 0 N. occidentalis 0 0 N. rustica 0 0 N. tabacum ' H a r r o w n o v a ' 0 0 N. tabacum ' S a r a s u n ' 0 0 N. tabacum ' X a n t h i ' 0 0 Petunia ' C o r a l S a t i n ' 0 0 Phaseolus vulgaris ' B l a c k T u r t l e ' 0 0 Vigna unguiculata 0 0 Zinnia elegans 0 0 a K e y : l i n e ; c h l = 1 1 = l o c a l l e s i o n s ; s y s = s y s t e m i c ; l e s = l e s i o n s ; l p = p a t t e r n ; d i s t = d i s t o r t i o n ; n e c = n e c r o t i c ; mo = m o t t l e ; : c h l o r o t i c ; 0 = no i n f e c t i o n F i g . 1 . Symptoms a s s o c i a t e d with apple c h l o r o t i c l e a f s p o t v i r u s ( i s o l a t e B) i n Prunus glandulosa (A) and Chenopodium quinoa (B) . c o n f i r m a t i o n by Koch's p o s t u l a t e s these symptoms must be desc r i b e d as ACLSV as s o c i a t e d . Various g r i n d i n g b u f f e r a d d i t i v e s a f f e c t e d the number of l o c a l l e s i o n s produced during rub tr a n s m i s s i o n t e s t s (Table 2 ) . The grea t e s t number of l e s i o n s occurred w i t h a d d i t i v e C (0.01M MgCl 2) . Without magnesium there was a complete l o s s of i n f e c t i v i t y a f t e r 24 hr. Over the range of pH 6.0 t o 8.5 there was no d i f f e r e n c e i n the s e v e r i t y or amount of systemic symptoms produced i n C. quinoa a f t e r rub i n o c u l a t i o n using TM b u f f e r . Likewise, d i l u t i o n of i n f e c t e d t i s s u e ground i n TM b u f f e r from 1:3 to 1:96 had no e f f e c t on systemic symptoms i n C. quinoa. A f t e r i n o c u l a t i o n w i t h i s o l a t e s A and B, no symptoms were found i n any of the greenhouse i n d i c a t o r s and a f t e r 1 year no symptoms were expressed i n the f i e l d i n d i c a t o r s . ACLSV clones from apple and pear used as p o s i t i v e checks produced t y p i c a l symptoms on P. veitchii, M. platycarpa, Spy 227, and R12740-7A but were not used on the Prunus i n d i c a t o r s . P o s i t i v e checks f o r other v i r u s e s normally detected by the Prunus i n d i c a t o r s induced t y p i c a l symptoms i n the appropriate hosts. 12 Table 2 . The e f f e c t s of b u f f e r a d d i t i v e s on P. glandulosa i s o l a t e B l o c a l l e s i o n number i n Chenopodium quinoa*. L o c a l l e s i o n number b I n o c u l a t e d I n o c u l a t e d A d d i t i v e 0 immediately a f t e r 24 hr A 66 0 B * d * d C 76 28 D 17 5 E 57 1 a T i s s u e was ground 1:3 (w/v) with the a p p r o p r i a t e a d d i t i v e , s t i r r e d 1 hr at 4 C, and c l a r i f i e d with the a d d i t i o n of b e n t o n i t e f o l l o w e d by a low speed c e n t r i f u g a t i o n . bAverage number of l e s i o n s per l e a f (12 leaves) CA11 b u f f e r s made with 0.01M T r i s pH 8.5 A = no a d d i t i v e s B = 0.01M MgCl 2, 0.15% sodium t h i o g l y c o l l a t e , 0.1% p e c t i n a s e , 3% T r i t o n X-100. C = 0.01M MgCl 2. D = 0.2% 3-3'-diaminodipropylamine. (DD) E = 0.01M MgCl 2, 0.1% p e c t i n a s e . dLeaves damaged by b u f f e r t o extent t h a t l e s i o n s c o u l d not be counted. 13 D i s c u s s i o n T h e h e r b a c e o u s h o s t r a n g e o f t h e A C L S V f r o m P. glandulosa a p p e a r s t o b e n a r r o w e r t h a n t h a t r e p o r t e d f r o m o t h e r woody p l a n t s . A n u m b e r o f h o s t s r e p o r t e d b y o t h e r a u t h o r s s u c h a s Beta vulgaris, Datura stramonium, Phaseolus vulgaris, Petunia hybrida, a n d Nicotiana tabacum ( 51 ,67 ) s h o w e d no symptoms when i n o c u l a t e d w i t h t h e A a n d B i s o l a t e s . T h e s e d i f f e r e n c e s do n o t a p p e a r t o b e u n i q u e t o Prunus s t r a i n s b e c a u s e t h e s t r a i n s u s e d f o r h o s t r a n g e s t u d i e s b y L i s t e r e t a l . (51) a n d b y S a k s e n a a n d M i n k (67) b o t h o r i g i n a t e d f r o m a p p l e y e t d i f f e r e d i n t h e i r h o s t r a n g e a n d s y m p t o m o l o g y . T h e P. glandulosa i s o l a t e h o s t r a n g e s w e r e i d e n t i c a l u s i n g p u r i f i e d v i r u s o r i n f e c t e d C. quinoa a s i n o c u l u m s o t h e e f f e c t s o f an i n h i b i t o r s u b s t a n c e f r o m C . quinoa d i d n o t i n f l u e n c e i n f e c t i v i t y . T h e A a n d B i s o l a t e s p r o d u c e d s l i g h t l y d i f f e r e n t symptoms on C. amaranticolor a n d C. murale ( T a b l e 1 ) . When t h e v i r u s was f i r s t i s o l a t e d f r o m t h e woody h o s t a n d p r e s e n t i n l o w c o n c e n t r a t i o n , o n l y l o c a l l e s i o n s w o u l d a p p e a r u n t i l a f t e r a few p a s s a g e s t h r o u g h C. quinoa. T h i s was n o t t h e c a s e f o r t h e A i s o l a t e , a s r e p e a t e d i n o c u l a t i o n s o f C. amaranticolor w i t h p u r i f i e d v i r u s o r i n f e c t e d C. quinoa n e v e r p r o d u c e d a n y s y s t e m i c s y m p t o m s a n d b a c k i n o c u l a t i o n s o f t h e s e s y m p t o m l e s s l e a v e s t o C. quinoa w e r e n e g a t i v e . T h i s l a c k o f s y s t e m i c i n v a s i o n i s i n c o n t r a s t t o t h e r e s u l t s r e p o r t e d i n t h e l i t e r a t u r e a n d t h e r e s u l t s o f t h e o t h e r A C L S V i s o l a t e s u s e d i n t h i s s t u d y , a l l o f w h i c h p r o d u c e d s y s t e m i c symptoms i n C. amaranticolor. The age o f i n o c u l a t e d C. quinoa a f f e c t e d t h e t y p e a n d s e v e r i t y o f symptoms e x p r e s s e d . T h i s i s e x p l a i n e d i n t e r m s o f t h e l o n g d i s t a n c e movement o f v i r u s e s t h r o u g h t h e p h l o e m t i s s u e i n a s s o c i a t i o n w i t h m e t a b o l i t e s . S a m u e l (70) s h o w e d t h a t TMV s p r e a d f r o m t h e i n o c u l a t e d l e a f , f i r s t t o t h e r o o t s , t h e n t h e y o u n g l e a v e s a n d m e r i s t e m , a n d f i n a l l y t h e o l d e r l e a v e s . T h e r e f o r e t h e amount o f t i m e f o r t h e v i r u s t o move s y s t e m i c a l l y i n a y o u n g p l a n t a n d t h e n u m b e r o f s y m p t o m l e s s l e a v e s b e t w e e n t h e i n o c u l a t i o n p o i n t a n d t h e g r o w i n g t i p i s l e s s t h a n i n an o l d e r p l a n t . F a c t o r s w h i c h i n f l u e n c e t h e c h o i c e o f e x t r a c t i o n b u f f e r f o r p u r i f i c a t i o n a r e p r e s e r v a t i o n o f p a r t i c l e i n t e g r i t y a n d i n f e c t i v i t y . M a g n e s i u m b e s t p r e s e r v e d i n f e c t i v i t y o v e r a 24 h r p e r i o d w h i l e p e c t i n a s e , w h i c h was a d d e d t o a i d i n t h e r e l e a s e o f p a r t i c l e s f r o m t h e p h l o e m t i s s u e , h a d a n e g a t i v e e f f e c t on i n f e c t i v i t y ( T a b l e 2 ) . D i v a l e n t c a t i o n s a r e r e q u i r e d b y v i r u s e s s u c h a s s o u t h e r n b e a n m o s a i c f o r s t a b i l i z i n g p r o t e i n - p r o t e i n i n t e r a c t i o n s ( 3 9 , 4 0 ) a n d m a g n e s i u m was a l s o e s s e n t i a l f o r s t a b i l i z i n g A C L S V d u r i n g p u r i f i c a t i o n ( 5 2 ) . T h e p o l y a m i n e DD, w h i c h h a s b e e n r e c o m m e n d e d as a s t a b i l i z i n g a g e n t (20) was n o t as e f f e c t i v e i n s t a b i l i z i n g t h e v i r u s as was m a g n e s i u m . T h e r e s u l t s o b t a i n e d f r o m woody i n d i c a t o r s w e r e u n e x p e c t e d b e c a u s e n o n e o f t h e m s h o w e d s y m p t o m s . B e s i d e s t h e one r e p o r t o f a " s u p e r - l a t e n t " s t r a i n w h i c h d i d n o t show on woody i n d i c a t o r s ( 3 1 ) , a l l o t h e r A C L S V i s o l a t e s h a v e p r o d u c e d symptoms on a t l e a s t o n e i n d i c a t o r c u l t i v a r . I n t h i s s t u d y two i m p o r t a n t A C L S V Prunus i n d i c a t o r s w e r e n o t a v a i l a b l e , n a m e l y GF 305 a n d P . tomentosa, w h i c h may h a v e d e t e c t e d t h e P. glandulosa i s o l a t e s . P. glandulosa i s u s u a l l y p r o p a g a t e d b y c u t t i n g s r a t h e r t h a n b u d d i n g o n u n d e r s t o c k , a l t h o u g h i t i s c o m p a t i b l e w i t h p e a c h a n d some p l u m r o o t s t o c k s . T h i s f a c t t o g e t h e r w i t h t h e s m a l l d i a m e t e r o f t h e s h o o t s may e x p l a i n t h e l a c k o f t r a n s m i s s i o n t o t h e woody i n d i c a t o r s o r t h e P. glandulosa i s o l a t e s may s i m p l y h a v e a v e r y n a r r o w h o s t r a n g e . C h a p t e r 3 - V i r u s P u r i f i c a t i o n I n t r o d u c t i o n A C L S V h a s b e e n p u r i f i e d b y a number o f w o r k e r s ( 5 1 , 6 9 , 5 2 , 2 0 , 4 , 4 9 , 5 0 ) . A l l o f t h e m e t h o d s h a v e i n common a c l a r i f i c a t i o n s t e p u s i n g b e n t o n i t e f o l l o w e d b y p o l y e t h y l e n e g l y c o l (PEG) p r e c i p i t a t i o n a n d / o r h i g h - s p e e d c e n t r i f u g a t i o n , t h e n r a t e z o n a l d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . T h e e f f e c t o f b e n t o n i t e o n v i r u s e s a n d p l a n t c o n s t i t u e n t s was d e t e r m i n e d b y D u n n a n d H i t c h b o r n ( 2 2 ) . R i b o s o m e s , 18 S p r o t e i n , g r e e n p i g m e n t s , a n d some v i r u s e s w e r e a b s o r b e d b y b e n t o n i t e w h i l e o t h e r v i r u s e s w e r e n o t a b s o r b e d . A C L S V i s v e r y l a b i l e a n d e a r l y p u r i f i c a t i o n a t t e m p t s r e s u l t e d i n h i g h l o s s e s due t o p a r t i c l e i n s t a b i l i t y a n d d e g r a d a t i o n . L i s t e r a n d H a d i d i (52) s h o w e d t h e s e l o s s e s c a n b e c o u n t e r a c t e d b y u s i n g m a g n e s i u m i n t h e e x t r a c t i o n b u f f e r t o s t a b i l i z e t h e p a r t i c l e , p r o b a b l y t h r o u g h i n c r e a s e d p r o t e i n -p r o t e i n i n t e r a c t i o n a n d b y i n a c t i v a t i o n o f r i b o n u c l e a s e a c t i v i t y w i t h b e n t o n i t e . C h a i r e z a n d L i s t e r (9) c o m p a r e d s t r a i n s o f A C L S V f r o m M a l u s a n d P r u n u s a n d f o u n d t h e y i e l d f r o m t h e P r u n u s s t r a i n s was o n e - f i f t h t h a t o f t h e a p p l e s t r a i n s a n d was n o t s i g n i f i c a n t l y i m p r o v e d w i t h m a g n e s i u m i n t h e e x t r a c t i o n b u f f e r . F o r t h e p l u m b a r k s p l i t s t r a i n , D u n e z e t a l . (20) f o u n d t h a t a h i g h pH ( 9 . 0 -9 .5 ) e x t r a c t i o n b u f f e r w i t h 0.2% 3 , 3 ' - d i a m i n o d i p r o p y l a m i n e (DD) was n e e d e d t o p r e v e n t p a r t i c l e a g g r e g a t i o n a n d i n s t a b i l i t y . T h i s s t r a i n was l e s s s t a b l e t h a n o n e s f r o m a p p l e a n d g a v e p o o r y i e l d s when p u r i f i e d b y t h e m e t h o d o f L i s t e r a n d H a d i d i . A m o d i f i c a t i o n was u s e d b y B a r - J o s e p h e t a l . (4) w h e r e b y v i r u s p u r i f i e d b y t h e 17 u s u a l m e t h o d was r u n on a C s S 0 4 i s o p y c n i c g r a d i e n t . I n C s S 0 4 g r a d i e n t s t h e v i r u s i s s t a b l e a n d d o e s n o t d e g r a d e a s i t d o e s i n C s C l . A n o t h e r v a r i a t i o n was u s e d b y L e g r a n d a n d V e r h o y e n (4 9 ) . T h e y h e l p e d p r e s e r v e p a r t i c l e i n t e g r i t y w i t h 0.25% f o r m a l d e h y d e a n d u s e d a s u c r o s e d e n s i t y g r a d i e n t s u p e r i m p o s e d on a r e v e r s e PEG g r a d i e n t t o s e p a r a t e t h e v i r u s . A p u r i f i c a t i o n m e t h o d w h i c h d i d n o t u s e b e n t o n i t e f o r c l a r i f i c a t i o n was d e s c r i b e d b y Thomas (72) u s i n g d i e t h y l e t h e r a n d c a r b o n t e t r a c h l o r i d e w h i c h g a v e a h i g h y i e l d (1 mg/100 g t i s s u e ) a n d A 2 6 0 / 2 8 0 r a t i o o f 1 . 7 2 . A p o i n t s t r e s s e d b y many a u t h o r s i s t h e v a r i a b l e amount o f b e n t o n i t e n e e d e d f o r c l a r i f i c a t i o n . I n t h e a b o v e m e n t i o n e d r e f e r e n c e s t h e q u a n t i t y v a r i e s a n d some a u t h o r s u s e a s i n g l e d o s e w h i l e o t h e r s p r e f e r m u l t i p l e d o s e s w i t h s m a l l e r v o l u m e s . The age a n d c o n d i t i o n o f t h e p l a n t h o s t a f f e c t s t h e amount o f b e n t o n i t e r e q u i r e d a n d e x c e s s b e n t o n i t e e l i m i n a t e s i n f e c t i v i t y ( 5 1 ) . A n u n u s u a l p r o p e r t y o f A C L S V i s t h e a b n o r m a l l y h i g h A 2 6 0 / 2 8 0 r a t i o ( 1 . 7 - 1 .8 ) c a u s e d b y a l a c k o f a r o m a t i c a m i n o a c i d s w h i c h a b s o r b i n t h e 280 nm r a n g e ( 4 ) . F a c t o r s w h i c h c a n l o w e r t h e r a t i o a r e c o n t a m i n a t i n g p r o t e i n s a n d p a r t i c l e b r e a k a g e . A n o t h e r p r o p e r t y o f A C L S V i s t h e d i f f i c u l t y i n s e p a r a t i n g t h e v i r u s f r o m h o s t m a t e r i a l . L i s t e r e t a l . (51) f o u n d two z o n e s o f h o s t o r i g i n i n t h e i r d e n s i t y g r a d i e n t t u b e s . Z o n e A , w h i c h r e s e m b l e d F r a c t i o n I p r o t e i n , was p r e s e n t i n h e a l t h y a n d i n f e c t e d s a m p l e s w h e r e a s z o n e B , c o n s i s t i n g o f p h y t o f e r r i t i n , was o n l y f o u n d i n i n f e c t e d s a m p l e s . O c c a s i o n a l l y , t h e r e m o v a l o f a l l p l a n t h o s t c o n s t i t u e n t s f r o m 18 a p u r i f i e d v i r u s p r e p a r a t i o n p r o v e s d i f f i c u l t w i t h s t a n d a r d m e t h o d s b e c a u s e t h e t r e a t m e n t s damage t h e v i r u s , t h e h o s t m a t e r i a l i s t i g h t l y b o u n d t o t h e v i r u s , o r t h e c o n t a m i n a n t h a s c h e m i c a l a n d p h y s i c a l p r o p e r t i e s s i m i l a r t o t h e v i r u s . A s p e c i f i c m e t h o d f o r e l i m i n a t i n g t h e s e c o n t a m i n a n t s i s t o p r e c i p i t a t e t h e m w i t h an a n t i s e r u m p r e p a r e d a g a i n s t h o s t a n t i g e n s ( 3 2 , 2 8 ) . T h e h o s t - a n t i b o d y c o m p l e x e s c a n t h e n b e r e m o v e d b y l o w - s p e e d c e n t r i f u g a t i o n . M a t e r i a l s a n d M e t h o d s T h e m o s t e f f e c t i v e a n d s i m p l e s t A C L S V p u r i f i c a t i o n m e t h o d was t o h o m o g e n i z e s y s t e m i c a l l y i n f e c t e d C . q u i n o a l e a v e s i n a W a r i n g b l e n d e r w i t h t h r e e v o l u m e s o f TM b u f f e r p l u s 0 . 0 2 M 2 -m e r c a p t o e t h a n o l (2-ME) a n d 15 m l / 1 0 0 g t i s s u e o f a b e n t o n i t e s u s p e n s i o n p r e p a r e d b y a v a r i a t i o n o f t h e m e t h o d d e s c r i b e d b y D u n n a n d H i t c h b o r n ( 2 2 ) . T w e n t y g r a m s o f b e n t o n i t e ( F i s h e r l a b g r a d e ) was h o m o g e n i z e d i n 400 m l o f 0 . 0 1 M T r i s p H 8 . 5 a n d c e n t r i f u g e d 1 m i n a t 400 g . T h e s u p e r n a t a n t was c e n t r i f u g e d 10 m i n a t 6000 g a n d t h e p e l l e t r e s u s p e n d e d i n 150 m l o f t h e same b u f f e r . T h i s p r o d u c e d a s u s p e n s i o n o f 5 0 - 5 5 m g / m l b e n t o n i t e . The h o m o g e n i z e d s a p was s q u e e z e d t h r o u g h c h e e s e c l o t h , s t i r r e d f o r 10 m i n a t 4 C , t h e n c e n t r i f u g e d f o r 15 m i n a t 9000 g . M o r e b e n t o n i t e (15 m l / 1 0 0 g) was a d d e d s l o w l y a n d t h e m i x t u r e s t i r r e d a n d c e n t r i f u g e d as a b o v e . The s u p e r n a t a n t was m i x e d w i t h 8.0% p o l y e t h y l e n e g l y c o l ( P E G ) , s t i r r e d 1 h r a t 4 C , t h e n c e n t r i f u g e d a t 1 4 , 5 0 0 g f o r 20 m i n . T h e p e l l e t s w e r e r e s u s p e n d e d i n 6 .0 m l t o t a l o f TM b u f f e r c o n t a i n i n g 1.0% T r i t o n X - 1 0 0 a n d s t i r r e d f o r 1-2 h r a t 4 C . C a e s i u m s u l p h a t e ( 3 . 5 g) was a d d e d a n d t h e s a m p l e b r o u g h t t o 10 m l w i t h TM b u f f e r , c e n t r i f u g e d 16 h r a t 1 7 1 , 0 0 0 g a n d t h e t h i n b a n d o f v i r u s was d r a w n o f f w i t h a n e e d l e a n d s y r i n g e . T h e s a m p l e was t h e n c e n t r i f u g e d f o r 90 m i n a t 1 3 8 , 0 0 0 g t h r o u g h a 1 . 5 m l 20% (w/v) s u c r o s e c u s h i o n made i n TM b u f f e r a n d t h e p e l l e t r e s u s p e n d e d i n a s m a l l v o l u m e o f TM b u f f e r f o r 16 h r o n a s h a k e r . V a r i o u s s t a g e s o f t h e p u r i f i c a t i o n a n d t h e f i n a l p r o d u c t w e r e e x a m i n e d i n t h e e l e c t r o n m i c r o s c o p e a n d s c a n n e d i n a s p e c t r o p h o t o m e t e r f r o m 2 3 0 - 3 6 0 nm. Some s a m p l e s w e r e a n a l y z e d f o r v i r u s c o n c e n t r a t i o n b y a B r a d f o r d p r o t e i n a s s a y ( B i o - R a d ) . A l s o , some s a m p l e s w e r e a n a l y z e d f o r p u r i t y b y s o d i u m d o d e c y l - s u l p h a t e 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 ( S D S - P A G E ) as d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n a n d some s a m p l e s w e r e t e s t e d b y d o u b l e a n t i b o d y s a n d w i c h E L I S A ( D A S - E L I S A ) f o r v i r u s c o n t e n t . To t e s t t h e r e l a t i v e a m o u n t s o f v i r u s l e f t i n t h e p e l l e t a n d s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n t h e p e l l e t s w e r e r e s u s p e n d e d i n an e q u a l v o l u m e o f b u f f e r a n d b o t h s a m p l e s a s s a y e d b y E L I S A . A s u c r o s e - P E G g r a d i e n t s i m i l a r t o t h a t u s e d b y L e g r a n d a n d V e r h o y e n (49) was t r i e d . A 5-30% s u c r o s e g r a d i e n t i n TM b u f f e r was s u p e r i m p o s e d on an i n v e r s e PEG g r a d i e n t b y p r e p a r i n g a 5% s u c r o s e s o l u t i o n w i t h 6% (w/v) P E G a n d a 30% s u c r o s e s o l u t i o n . T h e g r a d i e n t s w e r e p r e p a r e d w i t h a g r a d i e n t m a k e r w h i c h g r a d u a l l y m i x e s t h e d i l u t e s o l u t i o n i n t o t h e c o n c e n t r a t e d s o l u t i o n as t h e t u b e i s f i l l e d . T h e t u b e s w e r e l a y e r e d w i t h 200 / / l o f i s o l a t e B p u r i f i e d b y t h e s t a n d a r d m e t h o d a n d c e n t r i f u g e d 30 m i n a t 1 3 , 0 0 0 20 g . T h e t u b e s w e r e s c a n n e d a t 254 nm w i t h a g r a d i e n t f r a c t i o n a t o r ( I s c o ) . A s u c r o s e - c a e s i u m s t e p g r a d i e n t o f t h e t y p e u s e d f o r t h e p u r i f i c a t i o n o f o t h e r c l o s t e r o v i r u s e s (82) was a l s o t r i e d . C a e s i u m s u l p h a t e s o l u t i o n s o f 0 . 0 M , 0 . 4 M , 0 . 8 M , a n d 1 . 2 M w e r e p r e p a r e d w i t h 30% s u c r o s e (w/v) i n TM b u f f e r a n d 1 . 0 m l o f e a c h ( 1 . 2 M o n b o t t o m ) was l a y e r e d i n an SW 41 t u b e . R e s u s p e n d e d P E G p e l l e t s w e r e d i l u t e d t o 8 m l , l a y e r e d o v e r t h e s t e p g r a d i e n t a n d c e n t r i f u g e d f o r 90 m i n a t 1 7 5 , 0 0 0 g . T h e t u b e s w e r e t h e n s c a n n e d a t 254 nm o n a f r a c t i o n a t o r w i t h f l u o r i n e r t F C - 7 0 (Sigma) u s e d t o d i s p l a c e t h e g r a d i e n t f r o m t h e b o t t o m o f t h e t u b e . T h i s was f o l l o w e d b y c e n t r i f u g a t i o n i n t h e s t a n d a r d c a e s i u m s u l p h a t e i s o p y c n i c g r a d i e n t d e s c r i b e d e a r l i e r . T h e p u r i f i c a t i o n m e t h o d d e s c r i b e d b y L i s t e r a n d H a d i d i (52) was c o m p a r e d w i t h t h e e a r l i e r d e s c r i b e d s t a n d a r d m e t h o d . L i s t e r a n d H a d i d i ' s t e c h n i q u e was i d e n t i c a l up t o t h e r e s u s p e n s i o n o f t h e PEG p e l l e t s . T h e s a m p l e was t h e n c e n t r i f u g e d 5 m i n a t 2 , 0 0 0 g , t h e s u p e r n a t a n t was c e n t r i f u g e d 75 m i n a t 1 3 8 , 0 0 0 g a n d t h e p e l l e t r e s u s p e n d e d i n TM b u f f e r . A f t e r a l o w s p e e d c e n t r i f u g a t i o n o f 5 m i n a t 2 , 0 0 0 g t h e s u p e r n a t a n t was l a y e r e d o v e r a 10-40% s u c r o s e d e n s i t y g r a d i e n t a n d s p u n f o r 90 m i n a t 1 7 5 , 0 0 0 g . The t u b e s w e r e s c a n n e d a t 254 nm, t h e p e a k s c o l l e c t e d a n d c e n t r i f u g e d 75 m i n a t 138 , 000 g . The p e l l e t was r e s u s p e n d e d i n 250 o f TM b u f f e r . O t h e r v a r i a t i o n s o f t h e s t a n d a r d A C L S V p u r i f i c a t i o n m e t h o d w e r e t r i e d . T h e v i r u s was f i x e d i n 2% g l u t a r a l d e h y d e f o r 6 h r p r i o r t o c e n t r i f u g a t i o n i n c a e s i u m s u l p h a t e , a s e c o n d c a e s i u m s u l p h a t e g r a d i e n t was d o n e , t h e e x t r a c t i o n h o m o g e n a t e was s t i r r e d f o r 10 m i n a f t e r t h e a d d i t i o n o f t h e b e n t o n i t e , a P o l l a h n e r o l l e r p r e s s was c o m p a r e d t o a b l e n d o r f o r t i s s u e h o m o g e n a t i o n , a n d i n f e c t e d t i s s u e was f r o z e n i n l i q u i d n i t r o g e n p r i o r t o h o m o g e n a t i o n . O t h e r f a c t o r s i n v e s t i g a t e d w e r e t h e u s e o f two c l a r i f i c a t i o n s w i t h one h a l f b e n t o n i t e v o l u m e s e a c h v e r s u s one c l a r i f i c a t i o n w i t h f u l l v o l u m e b e n t o n i t e , t h e o m m i s i o n o f M g S 0 4 f r o m t h e e x t r a c t i o n b u f f e r , t h e u s e o f 20% v e r s u s 30% b e n t o n i t e f o r c l a r i f i c a t i o n , a t i s s u e t o b u f f e r r a t i o o f 1 : 4 . 5 ( w / v ) , t h e a d d i t i o n o f 1% N a C l d u r i n g PEG p r e c i p i t a t i o n , t h e age o f t h e i n f e c t e d t i s s u e , a n d a c o m p a r i s o n o f T r i t o n X - 1 0 0 a n d u r e a f o r r e s u s p e n s i o n o f t h e PEG p e l l e t s . To t e s t t h e e f f e c t s o f v a r i o u s f a c t o r s on A C L S V i n t e g r i t y , p u r i f i e d v i r u s was t r e a t e d as f o l l o w s a n d o b s e r v e d i n t h e e l e c t r o n m i c r o s c o p e . S a m p l e s w e r e d i l u t e d 1 : 5 w i t h e i t h e r pH 8 . 5 , 7 . 5 , 6 . 5 , o r 5 . 5 TM b u f f e r . O t h e r s a m p l e s w e r e d i l u t e d 1 : 5 w i t h e i t h e r 0 . 1 M , 0 . 0 5 M , 0 . 0 1 M , o r 0 . 0 0 5 M TM b u f f e r . V i r u s was a l s o d i l u t e d 1 : 5 i n TM b u f f e r w i t h e i t h e r 0.0%, 1.0%, 3.0%, o r 5.0% T r i t o n X - 1 0 0 . T h e e f f e c t o f 20% e t h a n o l , 8.0% b u t a n o l a n d 16.6% c h l o r o f o r m w e r e t e s t e d s e p a r a t e l y on p u r i f i e d v i r u s . H o s t a n t i g e n s w e r e p r e p a r e d b y h o m o g e n i z i n g C . q u i n o a t i s s u e i n two v o l u m e s 0 . 1 M b o r a t e p l u s 0 . 1 M E D T A p H 8, s q u e e z i n g t h r o u g h c h e e s e c l o t h a n d s t i r r i n g f o r 1 h r w i t h 2.0% T r i t o n X - 1 0 0 . The s a m p l e was c e n t r i f u g e d 20 m i n a t 1 4 , 5 0 0 g , t h e s u p e r n a t a n t s p u n f o r 90 m i n a t 1 1 1 , 0 0 0 g a n d t h e p e l l e t s r e s u s p e n d e d i n 4 . 0 m l b u f f e r . F o u r g r a m s o f c a e s i u m s u l p h a t e w e r e a d d e d a n d t h e m i x t u r e b r o u g h t t o 10 m l , t h e n c e n t r i f u g e d 16 h r a t 1 7 1 , 0 0 0 g . A n o p a l e s c e n t b a n d was e x t r a c t e d w i t h a n e e d l e a n d s y r i n g e a n d d i a l y s e d a g a i n s t t h e b o r a t e b u f f e r . The h o s t a n t i g e n s w e r e i n j e c t e d i n t r a m u s c u l a r l y i n t o a New Z e a l a n d W h i t e r a b b i t t o p r o d u c e a n t i s e r u m . F o u r h u n d r e d pil o f a n t i - h e a l t h y C . q u i n o a gamma g l o b u l i n s w e r e m i x e d w i t h 1 /5 v o l u m e (80/zl) P r o t e i n A / s e p h a r o s e C 1 - 4 B ( S i g m a ) , c e n t r i f u g e d 5 m i n a t 325 g , w a s h e d w i t h p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) , i n c u b a t e d 5 m i n a n d c e n t r i f u g e d a g a i n . F o u r h u n d r e d o f p u r i f i e d i s o l a t e A w e r e m i x e d w i t h t h e P r o t e i n - A / s e p h a r o s e g a m m a - g l o b u l i n p e l l e t , i n c u b a t e d 30 m i n a t room t e m p e r a t u r e ( R T ) , c e n t r i f u g e d a n d t h e s u p e r n a t a n t s a v e d . . T h e p e l l e t was w a s h e d w i t h P B S , i n c u b a t e d 5 m i n , c e n t r i f u g e d a n d t h e s u p e r n a t a n t s a v e d a g a i n w h i l e t h e p e l l e t was r e s u s p e n d e d i n 400 fj.1 P B S . A l l s a m p l e s w e r e t h e n a n a l y z e d b y e l e c t r o n m i c r o s c o p y , s p e c t r o p h o m e t e r a d s o r p t i o n s c a n , E L I S A a n d S D S - P A G E e l e c t r o p h o r e s i s . R e s u l t s T h e d e s c r i b e d A C L S V m e t h o d o f p u r i f i c a t i o n w o u l d t y p i c a l l y y i e l d 0 . 1 t o 0 . 2 5 mg v i r u s p e r 100 g o f t i s s u e , c a l c u l a t e d f r o m t h e UV a b s o r p t i o n s c a n u s i n g an e x t i n c t i o n c o e f f i c i e n t E 2 6 0 ( l m g / m l ) = 2 . 0 ( 4 ) . T h e A 2 6 0 / 2 8 0 r a t i o was 1 . 5 t o 1 . 7 ( c o r r e c t e d f o r l i g h t s c a t t e r i n g ) . T h e v i r u s c o n c e n t r a t i o n e s t i m a t e d f r o m t h e a b s o r p t i o n a t 260 nm was v e r y c l o s e ( 1 . 0 3 v e r s u s 0 . 9 m g / m l ) t o t h e c o n c e n t r a t i o n d e t e r m i n e d b y a B r a d f o r d p r o t e i n a s s a y . T h e h i g h 2 6 0 / 2 8 0 r a t i o was a g o o d e s t i m a t e o f p u r i t y a n d m i r r o r e d t h e r e s u l t s f r o m t h e e l e c t r o n m i c r o s c o p e a n d S D S - P A G E . S a m p l e s r u n on S D S - P a g e h a d a c o n t a m i n a t i n g h o s t b a n d a t MW 50 a n d 66 k i l o d a l t o n s ( k D a ) . T h e 50 kDa b a n d c o r r e s p o n d e d t o a h e a v y b a n d o f t h e same w e i g h t f o u n d i n h e a l t h y C . q u i n o a c l a r i f i e d s a p . T h e c r i t i c a l s t a g e i n t h e p u r i f i c a t i o n p r o c e d u r e was t h e b e n t o n i t e c l a r i f i c a t i o n . I f t h e s u p e r n a t a n t was l i g h t g r e e n r a t h e r t h a n a m b e r , t h e f i n a l p r o d u c t was c o n t a m i n a t e d d e s p i t e t h e u s e o f a n u m b e r o f t e c h n i q u e s t h a t w e r e i n t e n d e d t o e l i m i n a t e t h e h o s t m a t e r i a l . I f T r i t o n X - 1 0 0 (1.0%) was a d d e d t o t h e e x t r a c t i o n b u f f e r i n an e f f o r t t o b r e a k up h o s t m a t e r i a l a n d l i b e r a t e m o r e v i r i o n s , c l a r i f i c a t i o n became d i f f i c u l t . M o r e b e n t o n i t e was r e q u i r e d a n d b e c a u s e o f t h e g r e e n p i g m e n t s r e l e a s e d , i t was d i f f i c u l t t o a s s e s s t h e d e g r e e o f c l a r i f i c a t i o n . S i m i l a r r e s u l t s o c c u r r e d when 8% b u t a n o l o r 0.2% DD w e r e t r i e d i n t h e e x t r a c t i o n b u f f e r . C l a r i f i c a t i o n became d i f f i c u l t a n d a l t h o u g h t h e f i n a l p r o d u c t h a d more v i r u s , i t was i r r e v e r s i b l y c o n t a m i n a t e d t o t h e p o i n t w h e r e p u r i f i c a t i o n was n o t p o s s i b l e . A t t e m p t s t o s e p a r a t e h o s t c o m p o n e n t b y u s i n g a s e c o n d c a e s i u m g r a d i e n t , s u c r o s e d e n s i t y g r a d i e n t s (10-40% a n d 20 -50%) , a n d a s u c r o s e - P E G g r a d i e n t f a i l e d . A f t e r t h e c a e s i u m s u l p h a t e g r a d i e n t , t h e v i r u s was t y p i c a l l y c o n t a m i n a t e d w i t h h i g h m o l e c u l a r w e i g h t h o s t m a t e r i a l a n d many i r r e g u l a r s h a p e d s p h e r e s o f 10 -12 nm d i a m e t e r ( F i g . 2 ) . The m o s t e f f e c t i v e way t o e l i m i n a t e t h e s e s p h e r e s was a h i g h s p e e d c e n t r i f u g a t i o n t h r o u g h a 20% s u c r o s e c u s h i o n . T h e A 2 6 0 / 2 8 0 r a t i o m e a s u r e d b e f o r e a n d a f t e r t h i s 24 t r e a t m e n t was 0 . 8 6 and 1 . 2 r e s p e c t i v e l y and t h e sph e r e s were almost c o m p l e t e l y e l i m i n a t e d when viewed i n t h e e l e c t r o n m i c r o s c o p e . A s u c r o s e - c a e s i u m s t e p g r a d i e n t c o u l d be used t o e l i m i n a t e most o f t h e c o n t a m i n a t i n g spheres from a p u r i f i e d p r e p a r a t i o n but t h e v i r i o n s were fragmented a f t e r t h i s p r o c e d u r e . When t h e p r o c e d u r e was i n c o r p o r a t e d i n t o t h e s t a n d a r d one b e f o r e t h e i s o p y c n i c caesium g r a d i e n t no band was formed. When a s s a y e d by DAS-ELISA, t h e average l o s s o f v i r u s i n t h e f i r s t l o w - s p e e d c l a r i f i c a t i o n p e l l e t a v e r a g e d 28% ( r a n g e = 1 1 -38%) a n d t h e l o s s f o r t h e s e c o n d c l a r i f i c a t i o n s t e p a v e r a g e d 14% ( r a n g e = 5 - 2 4 % ) . E a r l i e r a t t e m p t s a t p u r i f i c a t i o n i n c l u d e d a l o w -s p e e d c e n t r i f u g a t i o n s t e p a f t e r t h e r e s u s p e n s i o n o f t h e PEG p e l l e t s a n d a t t h i s s t a g e t h e a v e r a g e l o s s o f v i r u s i n t h e p e l l e t a v e r a g e d 54% ( r a n g e = 37-64%) e v e n i f t h e s p e e d a n d t i m e w e r e r e d u c e d t o 5 m i n a t 2000 g . T h e m e t h o d o f L i s t e r a n d H a d i d i (52) c o u l d b e u s e d t o p u r i f y A C L S V p r o v i d e d t h e t i s s u e was c l a r i f i e d s u f f i c i e n t l y . T h e y i e l d a n d p u r i t y o f v i r u s f r o m t h i s m e t h o d w e r e e q u a l t o t h e m e t h o d d e s c r i b e d i n t h i s p a p e r when a s s a y e d b y S D S - P A G E a n d e l e c t r o n m i c r o s c o p y . T h e f o l l o w i n g a d d i t i o n a l v a r i a b l e s w e r e i n v e s t i g a t e d f o r t h e i r e f f e c t o n v i r u s y i e l d a n d c o n t a m i n a t i o n d u r i n g p u r i f i c a t i o n . When t h e v i r u s was f i x e d w i t h 2% g l u t a r a l d e h y d e f o r 6 h r p r i o r t o c e n t r i f u g a t i o n i n c a e s i u m s u l p h a t e , no b a n d was f o r m e d . A f t e r a s e c o n d c a e s i u m s u l p h a t e g r a d i e n t t h e v i r a l b a n d was much s m a l l e r t h a n t h e f i r s t b a n d a n d e q u a l l y c o n t a m i n a t e d . C l a r i f i c a t i o n was i m p r o v e d b y s t i r r i n g a f t e r t h e a d d i t i o n o f b e n t o n i t e f o r 10 m i n a t 4 C . H o s t m a t e r i a l was r e d u c e d w i t h no l o s s o f v i r u s . I n a c o m p a r i s o n b e t w e e n a P o l l a h n e r o l l e r p r e s s a n d a b l e n d e r f o r t i s s u e h o m o g e n i z a t i o n t h e r e was no d i f f e r e n c e i n p a r t i c l e f r a g m e n t a t i o n a n d t h e r o l l e r p r e s s s a m p l e was s l i g h t l y more c o n t a m i n a t e d . I f t i s s u e was p o w d e r e d i n l i q u i d n i t r o g e n i n a c o f f e e m i l l p r i o r t o b l e n d i n g , no v i r u s b a n d was f o r m e d . The u s e o f two c l a r i f i c a t i o n s w i t h one h a l f b e n t o n i t e v o l u m e s e a c h v e r s u s one c l a r i f i c a t i o n w i t h f u l l v o l u m e i n c r e a s e d v i r u s y i e l d ( 0 . 3 5 t o 0 . 4 1 m g / m l ) , a n d h o s t c o n t a m i n a t i o n , as j u d g e d i n t h e e l e c t r o n m i c r o s c o p e , was s l i g h t l y l e s s . The o m i s s i o n o f M g S 0 4 f r o m , t h e e x t r a c t i o n b u f f e r r e s u l t e d i n p a r t i a l p a r t i c l e d e g r a d a t i o n . When 20% b e n t o n i t e (20 m l / 1 0 0 g t i s s u e ) was u s e d r a t h e r t h a n t h e s t a n d a r d 30% t h e y i e l d was i n c r e a s e d ( 0 . 3 2 m g / m l t o 1 . 0 5 m g / m l ) b u t t h e s a m p l e was more c o n t a m i n a t e d . A t i s s u e t o b u f f e r r a t i o o f 1 : 4 . 5 (w/v) d i d ' n o t i m p r o v e t h e y i e l d o r r e d u c e c o n t a m i n a t i o n . T h e a d d i t i o n o f 1% N a C l d u r i n g PEG p r e c i p i t a t i o n i n c r e a s e d c o n t a m i n a t i o n ( A 2 6 0 / 2 8 0 1 . 2 v e r s u s 1 .7 ) a n d a l s o i n c r e a s e d v i r u s y i e l d ( 0 . 9 1 v e r s u s 0 . 4 6 m g / m l ) . T w e n t y d a y o l d t i s s u e was l e s s c o n t a m i n a t e d , e s p e c i a l l y w i t h s p h e r e s , t h a n 35 d a y t i s s u e . U r e a (0 .5M) was n o t b e t t e r t h a n T r i t o n X - 1 0 0 (1.0%) f o r d e c r e a s i n g p a r t i c l e a g g r e g a t i o n d u r i n g r e s u s p e n s i o n o f t h e P E G p e l l e t s . V a r i o u s t r e a t m e n t s w e r e t r i e d t o b r e a k up t h e a g g r e g a t i o n s o f h o s t a n d v i r u s i n a c o n t a m i n a t e d p u r i f i e d p r e p a r a t i o n . T h e pH o f t h e TM b u f f e r h a d an e f f e c t on v i r u s s t a b i l i t y . When p u r i f i e d i s o l a t e A was d i l u t e d (1 :5 ) w i t h pH 7 . 5 , 6 . 5 , a n d 5 . 5 b u f f e r , t h e r o d s w e r e d e g r a d e d when v i e w e d i n t h e e l e c t r o n m i c r o s c o p e w h i l e t h e u n t r e a t e d v i r u s r e m a i n e d i n t a c t . T h e i o n i c s t r e n g t h o f t h e pH 8 . 5 TM b u f f e r h a d no e f f e c t on v i r a l s t a b i l i t y o v e r t h e r a n g e 0 . 1 M t o 0 . 0 0 5 M . One p e r c e n t a n d 3.0% T r i t o n X - 1 0 0 h a d no e f f e c t on v i r a l s t a b i l i t y o r c l u m p i n g o f v i r u s a n d h o s t , h o w e v e r , when 5% was u s e d t h e c l u m p s o f v i r u s a n d h o s t w e r e b r o k e n u p b u t t h e v i r u s was p a r t i a l l y d e g r a d e d . T w e n t y p e r c e n t e t h a n o l h a d no e f f e c t on t h e v i r u s w h e r e a s 8% b u t a n o l a n d 16.6% c h l o r o f o r m g a v e 27 r e s u l t s s i m i l a r t o 5.0% T r i t o n X - 1 0 0 w h e r e b o t h t h e v i r u s a n d h o s t w e r e d e g r a d e d . E v e n w i t h t h e o p t i m u m A C L S V p u r i f i c a t i o n p r o c e d u r e s m a l l a m o u n t s o f h o s t m a t e r i a l s t i l l r e m a i n e d . T h e a n t i s e r u m a g a i n s t h e a l t h y C . q u i n o a r e a c t e d a g a i n s t f i v e o t h e r s p e c i e s ( N . g l u t i n o s a , N . b e n t h a m n i a n a , C . a m a r a n t i c o l o r , V . u n g u i c u l a t a , a n d C . s a t i v a ) i n a g a r g e l d o u b l e d i f f u s i o n t e s t s i n d i c a t i n g t h e a n t i s e r u m d e t e c t s one o r more c o m p o n e n t s common t o a n u m b e r o f d i f f e r e n t p l a n t s . A f t e r p u r i f i e d i s o l a t e A v i r u s was c r o s s -a d s o r b e d w i t h a n t i - h o s t a n t i s e r u m , t h e two c o n t a m i n a t i n g b a n d s o b s e r v e d i n t h e p r e - a d s o r b e d p r e p a r a t i o n i n S D S - P A G E w e r e s t i l l p r e s e n t a n d i n t h e e l e c t r o n m i c r o s c o p e t h e two s a m p l e s a p p e a r e d i d e n t i c a l . T h e UV a b s o r p t i o n s c a n o f b o t h p r e p a r a t i o n s s h o w e d t h e v i r u s c o n c e n t r a t i o n d r o p p e d f r o m 0 . 3 6 5 m g / m l t o 0 . 2 7 8 m g / m l a f t e r c r o s s a d s o r p t i o n . T h e p e l l e t f r o m t h e c r o s s a d s o r p t i o n s t e p was r e s u s p e n d e d a n d c e n t r i f u g e d a g a i n a n d t h e s u p e r n a t a n t a n d p e l l e t e x a m i n e d b y E L I S A a n d S D S - P A G E f o r e v i d e n c e o f n o n - s p e c i f i c a n d s p e c i f i c b i n d i n g o f v i r u s . By E L I S A t h e A 4 0 5 f o r t h e c r o s s a d s o r b e d s u p e r n a t a n t was 0 . 3 8 8 , t h e p e l l e t s u p e r n a t a n t was 0 . 1 6 6 a n d t h e r e s u s p e n d e d p e l l e t was 0 . 0 6 . I n S D S - P A G E a s m a l l v i r u s b a n d was s e e n i n t h e p e l l e t s u p e r n a t a n t a n d a s m a l l e r b a n d i n t h e r e s u s p e n d e d p e l l e t ( F i g . 3 ) . T h e s e d a t a i n d i c a t e t h a t t h e c o n t a m i n a t i n g h o s t c o m p o n e n t c o u l d n o t b e r e m o v e d b y c r o s s -a d s o r p t i o n a n d t h a t a s m a l l amount o f v i r u s was l o s t b y s p e c i f i c a n d n o n - s p e c i f i c b i n d i n g t o t h e P r o t e i n A/gamma g l o b u l i n s e p h a r o s e b e a d . F i g . 3 . S D S - P A G E o f c r o s s - a d s o r p t i o n ( C / A ) o f i s o l a t e A . L a n e s A a n d H = l o w MW s t a n d a r d s ( B i o - R a d ) . B = p r e - C / A v i r u s . C = s u p e r n a t a n t f r o m C / A . D = s u p e r n a t a n t f r o m p e l l e t c e n t r i f u g a t i o n . E = p e l l e t f r o m p e l l e t c e n t r i f u g a t i o n . F = A C L S V m o n o c l o n a l g a m m a - g l o b u l i n . G = a n t i - h o s t g a m m a - g l o b u l i n . N o t e c o n t a m i n a t i n g b a n d s a t 50 a n d 66 KDa i n l a n e s C a n d D a n d v i r u s b a n d a t 26 KDa i n l a n e s D a n d E . D i s c u s s i o n The d e s c r i b e d A C L S V p u r i f i c a t i o n m e t h o d i s s i m p l e a n d q u i c k , a n d g i v e s a p r o d u c t o f e q u a l c o n c e n t r a t i o n a n d p u r i t y when 29 c o m p a r e d t o t h e s t a n d a r d m e t h o d o f L i s t e r a n d H a d i d i ( 5 2 ) . I t i s a l s o e a s i e r t o u s e b e c a u s e h o m o g e n a t e s t h a t a r e n o t s u f f i c i e n t l y c l a r i f i e d w i l l s t i l l y i e l d v i r u s . I f t h e c l a r i f i c a t i o n was p o o r , a t t e m p t s t o p u r i f y A C L S V b y t h e s t a n d a r d m e t h o d f a i l e d b e c a u s e o f v i r u s l o s s i n l o w - s p e e d a n d d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n t h r o u g h p a r t i c l e a g g r e g a t i o n . T h e d e s c r i b e d m e t h o d a v o i d s l o w -s p e e d c e n t r i f u g a t i o n a f t e r t h e i n i t i a l c l a r i f i c a t i o n . L o s s e s d u r i n g c l a r i f i c a t i o n f r o m v i r i o n s a g g r e g a t i n g w i t h h o s t t i s s u e a r e s u b s t a n t i a l . Many m e t h o d s d e s i g n e d t o l i b e r a t e m o r e v i r i o n s b y b r e a k i n g u p a g g r e g a t e s o f v i r u s a n d h o s t w e r e u n s u c c e s s f u l r e s u l t i n g i n more c o n t a m i n a t i o n o r b r e a k d o w n o f v i r u s . A n a r e a w o r t h y o f f u r t h e r r e s e a r c h i s how t o a v o i d v i r u s l o s s d u r i n g c l a r i f i c a t i o n a n d t o r e d u c e . c o n t a m i n a t i o n , a common c o n s e q u e n c e o f i n c r e a s i n g v i r u s y i e l d . A n u n e x p e c t e d r e s u l t was t h e f a i l u r e o f t h e p o l y a m i n e DD t o i m p r o v e p u r i f i c a t i o n s i n c e D u n e z e t a l . (20) f o u n d DD t o b e e s s e n t i a l f o r p u r i f y i n g t h e b a r k - s p l i t s t r a i n o f A C L S V f r o m P r u n u s d o m e s t i c a b y p r e v e n t i n g a g g r e g a t i o n o f t h e v i r u s w i t h r i b o s o m a l s u b u n i t s . I t may n o t b e p o s s i b l e t o a s s u m e t h a t a l l A C L S V P r u n u s s t r a i n s b e h a v e t h e same d u r i n g p u r i f i c a t i o n . T h e p l a n t c o n s t i t u e n t s m o s t c o m m o n l y f o u n d c o n t a m i n a t i n g p u r i f i e d v i r u s p r e p a r a t i o n s a r e F r a c t i o n I p r o t e i n ( r i b u l o s e d i p h o s p h a t e c a r b o x y l a s e ) , p h y t o f e r r i t i n , a n d r i b o s o m a l s u b u n i t s ( 2 9 ) . A l l o f t h e s e h a v e a d i a m e t e r o f a p p r o x i m a t e l y 1 0 - 1 2 nm. The MW o f F r a c t i o n I p r o t e i n s u b u n i t s i s 55 a n d 12 KDa ( 4 3 ) , w h i l e p h y t o f e r r i t i n i s c o m p o s e d o f 28 a n d 2 6 . 5 KDa p o l y p e p t i d e s ( 4 8 ) . 30 T h e i d e n t i t y o f t h e c o n t a m i n a t i n g s p h e r e s i n my p r e p a r a t i o n s i s u n k n o w n . U n l i k e p h y t o f e r r i t i n , t h e s e s p h e r e s d i d n o t c o n t a i n an e l e c t r o n - d e n s e c o r e when u n s t a i n e d p r e p a r a t i o n s w e r e v i e w e d i n t h e e l e c t r o n m i c r o s c o p e , h o w e v e r , t h e c o n c e n t r a t i o n o f s p h e r e s i n c r e a s e d i n o l d e r p l a n t s , a c h a r a c t e r i s t i c o f p h y t o f e r r i t i n a s s o c i a t e d w i t h i t ' s r e g u l a t i o n o f e x c e s s i r o n r e l e a s e d f r o m c h l o r o p l a s t b r e a k d o w n . The MW o f F r a c t i o n I p r o t e i n r e s e m b l e s t h a t o f t h e s p h e r e s , h o w e v e r , t h e c o n c e n t r a t i o n o f t h i s enzyme s h o u l d n o t i n c r e a s e i n o l d e r p l a n t s . T h e o t h e r p o s s i b l e i d e n t i t y f o r t h e c o n t a m i n a t i n g b a n d s may b e one o f t h e r i b o s o m a l p r o t e i n s a s r e p o r t e d b y D u n e z ( 2 0 ) . I t was h o p e d t h a t a m e t h o d c o u l d b e d e v i s e d t o s e p a r a t e h o s t c o n s t i t u e n t s a n d v i r u s i n p a r t i a l l y p u r i f i e d p r e p a r a t i o n s b u t a n u m b e r o f t h e s e m e t h o d s f a i l e d . T h e e f f e c t s o f l o w p H TM b u f f e r o n p u r i f i e d v i r u s was u n u s u a l . A t l o w pH t h e n e e d f o r m a g n e s i u m i o n s t o s t a b i l i z e A C L S V s t r u c t u r e i s r e d u c e d a s i t i s w i t h s p h e r i c a l v i r u s e s d e p e n d a n t on d i v a l e n t c a t i o n s f o r s t a b i l i z a t i o n ( 5 2 ) . T h e f a c t t h a t l o w pH TM b u f f e r p a r t i a l l y d i s r u p t e d t h e p a r t i c l e s i s e v e n more u n u s u a l c o n s i d e r i n g i n f e c t i v i t y was n o t a f f e c t e d when i n f e c t e d C . q u i n o a was g r o u n d i n t h e s e b u f f e r s . A p H d e p e n d a n t i n t e r a c t i o n b e t w e e n t h e v i r u s a n d t h e u r a n y l a c e t a t e s t a i n may b e r e s p o n s i b l e f o r t h e d e c r e a s e d p a r t i c l e s t a b i l i t y . C o n c e n t r a t i o n s o f T r i t o n X - 1 0 0 i n t h e 3-5% r a n g e h a v e b e e n u s e d i n p u r i f y i n g g r a p e v i n e l e a f r o l l (84) b u t t h e s e l e v e l s a r e d e t r i m e n t a l t o A C L S V . T h e f a i l u r e o f t h e c r o s s - a d s o r p t i o n w i t h a n t i - h o s t a n t i s e r u m b o u n d t o s e p h a r o s e b e a d s was e x p e c t e d i n v i e w o f t h e A C L S V m o r p h o l o g y . I t i s e a s y t o i m a g i n e how l o n g f l e x u o u s r o d s w o u l d b e c o m e e n t a n g l e d on t h e s e p h a r o s e b e a d s a n d b e l o s t i n t h e c e n t r i f u g a t i o n s t e p . When t h e p e l l e t f r o m t h i s s t e p was r e s u s p e n d e d a n d c e n t r i f u g e d a g a i n , b o t h t h e s u p e r n a t a n t a n d p e l l e t c o n t a i n e d v i r u s w h i c h i n d i c a t e s t h a t a t l e a s t some o f t h e v i r u s was n o n - s p e c i f i c a l l y b o u n d t o t h e b e a d s a n d was r e l e a s e d d u r i n g r e s u s p e n s i o n o f t h e p e l l e t . T h e v i r u s r e m a i n i n g i n t h e p e l l e t was e i t h e r s p e c i f i c a l l y b o u n d t o t h e a n t i - h o s t i m m u n o g l o b u l i n s o r b e c a u s e o f i t s p h y s i c a l n a t u r e was n o t r e l e a s e d d u r i n g r e s u s p e n s i o n . T h i s l o s s o f v i r u s c o u l d b e j u s t i f i e d i f c r o s s - a d s o r p t i o n w o r k e d , b u t t h e c o n t a m i n a t i n g b a n d s ( F i g . 3) w e r e n o t e l i m i n a t e d . F o r an e f f e c t i v e c r o s s - a d s o r p t i o n t e c h n i q u e t o b e d e v e l o p e d f o r A C L S V , a more s p e c i f i c a n t i - h o s t g a m m a - g l o b u l i n i s n e e d e d i n c o n j u n c t i o n w i t h a m e t h o d w h i c h w o u l d a v o i d t h e n o n - s p e c i f i c b i n d i n g o f t h e v i r u s , p o s s i b l y a n i m m u n o p r e c i p i t a t i n g g e l f i l t r a t i o n c o l u m n . 32 C h a p t e r 4 - C v t o p a t h o l o g y a n d P h y s i c a l P r o p e r t i e s  I n t r o d u c t i o n C l o s t e r o v i r u s e s show a g r e a t d i v e r s i t y i n f e a t u r e s s u c h as c y t o p a t h o l o g y a n d v e c t o r r e l a t i o n s h i p s a n d h a v e t h e r e f o r e b e e n p l a c e d i n t o t h r e e s u b g r o u p s ( 5 ) . S u b g r o u p A h a s p a r t i c l e s 700 -800 nm, s u b g r o u p B 1200 - 1500 nm a n d s u b g r o u p C 1500 - 2000 nm. P a t h o l o g i c a l c h a n g e s f o u n d i n s u b g r o u p s B a n d C i n c l u d e v i r u s , p a r t i c l e s i n l a r g e m a s s e s , u s u a l l y i n t h e p h l o e m o r p h l o e m p a r e n c h y m a a n d c y t o p l a s m i c membranous v e s i c l e s ( 2 5 ) . O t h e r g e n e r a l n e c r o g e n i c f e a t u r e s s u c h as c h l o r o p l a s t a n d m i t o c h o n d r i a d i s i n t e g r a t i o n a n d a c c u m u l a t i o n o f o s m i o p h i l i c g l o b u l e s a n d p h y t o f e r r i t i n i n t h e c h l o r o p l a s t a r e f o u n d i n a l l s u b g r o u p s . T h e b e s t s t u d i e d c l o s t e r o v i r u s i s t h e t y p e member , b e e t y e l l o w s v i r u s ( B Y V ) , w h i c h f o r m s l a r g e m a s s e s o f p a r t i c l e s o f t e n a s s o c i a t e d w i t h m e m b r a n o u s v e s i c l e s r e f e r r e d t o as " B Y V - t y p e " v e s i c l e s ( 2 3 ) . T h e s e v e s i c l e s a r e a l s o a s s o c i a t e d w i t h a n u m b e r o f o t h e r c l o s t e r o v i r u s e s . I n c o n t r a s t t o t h e m a j o r i t y o f c l o s t e r o v i r u s e s w h i c h a r e f o u n d o n l y i n t h e v a s c u l a r e l e m e n t s , O h k i e t a l . (61) f o u n d A C L S V , a p p l e s t e m g r o o v i n g v i r u s a n d c i t r u s t a t t e r l e a f v i r u s i n t h e m e s o p h y l l a n d p h l o e m p a r e n c h y m a c e l l s , b u t n o t t h e e p i d e r m i s o r s i e v e e l e m e n t s o f i n f e c t e d C . q u i n o a . A C L S V p a r t i c l e s w e r e o b s e r v e d as r o u n d a g g r e g a t i o n s o r a s c r y s t a l l i z e d p a r a l l e l o g r a m s i n t h e c y t o p l a s m a n d n u c l e u s . T h e p h y s i c a l p r o p e r t i e s o f A C L S V h a v e b e e n r e p o r t e d p r e v i o u s l y ( 4 , 7 2 , 8 1 , ) . A n a l y s i s o f A C L S V a n d o t h e r c l o s t e r o v i r u s d s R N A r e p l i c a t i v e f o r m s b y PAGE g a v e p a t t e r n s t h a t w e r e d i s t i n c t f o r 33 e a c h v i r u s b u t , w i t h t h e e x c e p t i o n o f A C L S V , s i m i l a r e n o u g h t o b e d i a g n o s t i c f o r c l o s t e r o v i r u s e s ( 1 9 ) . M a t e r i a l s a n d M e t h o d s L e a f t i s s u e f r o m P . g l a n d u l o s a a n d C . q u i n o a was h a r v e s t e d f r o m s y s t e m i c a l l y i n f e c t e d g r e e n h o u s e g r o w n p l a n t s i n J u n e . S m a l l p i e c e s o f p e t i o l e , m i d r i b a n d l e a f b l a d e w e r e f i x e d o v e r n i g h t (0 /N) i n 4% g l u t a r a l d e h y d e i n pH 7, 0 . 1 M c a c o d y l a t e b u f f e r , w a s h e d t w i c e w i t h c a c o d y l a t e b u f f e r a n d l e f t i n 1% 0 s 0 4 i n pH 7, 0 . 1 M c a c o d y l a t e b u f f e r f o r 1 h r . S a m p l e s w e r e p u t t h r o u g h a d e h y d r a t i o n s e r i e s o f two w a s h e s e a c h i n 50%, 70%, 95% a n d a b s o l u t e e t h a n o l (EtOH) a n d t h e n w a s h e d t h r e e t i m e s i n p r o p y l e n e o x i d e b e f o r e b e i n g i n f i l t r a t e d O / N i n a 5 0 / 5 0 m i x t u r e o f p r o p y l e n e o x i d e a n d E p o n 8 1 2 . F i n a l l y , t h e s a m p l e s w e r e t r a n s f e r r e d t o e m b e d d i n g m o l d s f i l l e d w i t h f r e s h E p o n . T h e e m b e d d e d s a m p l e s w e r e s e c t i o n e d on a R e i c h e r t Om U2 m i c r o t o m e w i t h g l a s s k n i v e s t o o b t a i n s e c t i o n s w i t h g o l d a n d s i l v e r d i f f r a c t i o n c o l o u r s w h i c h w e r e t r a n s f e r r e d t o 100 m e s h c o p p e r g r i d s . S e c t i o n s w e r e s t a i n e d w i t h 4% a q u e o u s u r a n y l a c e t a t e ( U A ) , w a s h e d w i t h d i s t i l l e d w a t e r (dH 2 0) , s t a i n e d w i t h R e y n o l d s l e a d c i t r a t e d i l u t e d 1 :4 i n 0 . 0 1 N N a O H , w a s h e d a g a i n a n d b l o t t e d d r y . F o u r h u n d r e d mesh c o p p e r g r i d s w e r e f l o a t e d on d r o p s o f v i r u s p u r i f i e d f r o m C . q u i n o a ( v i r u s c o n c e n t r a t i o n 0 . 5 t o 1 . 0 m g / m l ) i n pH 8, 0 . 0 1 M T r i s b u f f e r w i t h 0 . 0 1 M M g S 0 4 , r i n s e d on a d r o p o f d H 2 0 a n d s t a i n e d w i t h 8 d r o p s o f 2% a q u e o u s UA o r p h o s p h o t u n g s t i c a c i d ( P T A ) . A l l m i c r o g r a p h s w e r e t a k e n o n an H i t a c h i 600 e l e c t r o n m i c r o s c o p e a t 75 K v a c c e l e r a t i n g v o l t a g e . T h e v i r u s m o d a l l e n g t h s w e r e c a l c u l a t e d b y m e a s u r i n g n o n - f r a g m e n t e d p a r t i c l e s o n m i c r o g r a p h s w i t h a c a l i b r a t e d t r a c i n g w h e e l a t f i n a l m a g n i f i c a t i o n s o f 3 7 , 5 0 0 t o 150,000. M o d a l l e n g t h s w e r e d e t e r m i n e d b y t a k i n g t h e mean o f t h e m o s t f r e q u e n t h i s t o g r a m c e l l a n d t h e c o n t i g u o u s c e l l s . M e a s u r e m e n t s o f v i r u s w i d t h a n d p i t c h w e r e made f r o m m i c r o g r a p h s w i t h a f i n a l m a g n i f i c a t i o n o f 1 5 0 , 0 0 0 t o 375,000 u s i n g a L e i t z 8x m a g n i f i e r . T h e m i c r o s c o p e was c a l i b r a t e d a t e a c h m a g n i f i c a t i o n u s i n g a 2160 l i n e s p e r cm d i f f r a c t i o n g r a t i n g . V i r a l c o a t p r o t e i n m o l e c u l a r w e i g h t s w e r e d e t e r m i n e d b y d i s c o n t i n u o u s 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 w i t h a 4% s t a c k i n g g e l a n d a 12% o r 15% r u n n i n g g e l i n a m i n i - v e r t i c a l s l a b g e l a p p a r a t u s ( B i o - R a d ) w i t h t h e b u f f e r s y s t e m o f L a e m m l i ( 4 7 ) . S a m p l e s w e r e b o i l e d f o r 4 m i n i n 0 . 0 6 M T r i s , p H 6 .8 w i t h 10% g l y c e r o l , 2% s o d i u m d o d e c y l s u l p h a t e ( S D S ) , 5% 2 - M E a n d 0.00125% b r o m o p h e n o l b l u e b e f o r e l o a d i n g o n t h e g e l . S t a n d a r d s w e r e B i o -R a d l o w m o l e c u l a r w e i g h t s t a n d a r d s (92 t o 1 4 . 4 K D a ) . T h e a m p e r a g e was s e t a t 10 m A / g e l u n t i l t h e s a m p l e s r e a c h e d t h e r u n n i n g g e l , t h e n 25 m A / g e l u n t i l t h e t r a c k i n g d y e r e a c h e d t h e b o t t o m o f t h e g e l . T h e g e l s w e r e s t a i n e d w i t h C o o m a s s i e B r i l l i a n t B l u e R - 2 5 0 i n 25% m e t h a n o l a n d 70% a c e t i c a c i d f o r 1 h r t h e n d e s t a i n e d O / N i n 25% m e t h a n o l , 7% a c e t i c a c i d . F o r w e s t e r n b l o t a n a l y s i s g e l s w e r e n o t s t a i n e d b u t s o a k e d i n b l o t t i n g b u f f e r (25 mM T r i s , 192 mM g l y c i n e , 20% m e t h a n o l , pH 8 .3 ) f o r 30 m i n a n d t r a n s f e r r e d t o an I m m o b i l o n membrane 35 ( M i l l i p o r e ) i n a B i o - R a d t r a n s - b l o t c e l l f o r 1 . 5 h r a t 100 V a t 4 C . T h e b l o t s w e r e b l o c k e d 0 / N i n p h o s p h a t e b u f f e r e d s a l i n e (PBS) w i t h 0.05% T w e e n - 2 0 (PBS-Tw) a n d 1% n o n - f a t m i l k p o w d e r ( B l o t t o ) ( 4 1 ) , w a s h e d t h r e e t i m e s i n P B S - T w a n d s o a k e d i n p o l y c l o n a l a n t i s e r u m d i l u t e d 1 : 5 0 0 w i t h c l a r i f i e d C . q u i n o a s a p f o r 2 h r a t r o o m t e m p e r a t u r e ( R T ) . T h e b l o t s w e r e w a s h e d t h r e e t i m e s w i t h P B S - T w t h e n s o a k e d 1 . 5 h r i n 1 2 5 i - l a b e l l e d P r o t e i n - A p r e p a r e d a c c o r d i n g t o B o l t o n a n d H u n t e r ( 6 ) . T h e b l o t s w e r e t h e n w a s h e d f o u r t i m e s i n P B S - T w , b l o t t e d d r y a n d a u t o r a d i o g r a p h e d O / N a t -70 C w i t h i n t e n s i f y i n g s c r e e n s . F o r RNA e x t r a c t i o n 500 o f p u r i f i e d i s o l a t e A was m i x e d w i t h 100 fj.1 0 . 0 5 M T r i s p H 8 . 9 , 25 fj.1 20% S D S , 200 /xl c h l o r o f o r m -i s o a m y l a l c o h o l (24 :1 ) a n d 200 r e d i s t i l l e d p h e n o l s a t u r a t e d w i t h d H 2 0 . A f t e r a l o w s p e e d c e n t r i f u g a t i o n t h e a q u e o u s p h a s e was e x t r a c t e d w i t h 300 fj.1 p h e n o l a n d 300 fj.1 c h l o r o f o r m - i s o a m y l a l c o h o l a n d t h e n w i t h 600 fj.1 c h l o r o f o r m - i s o a m y l a l c o h o l . T h e RNA was p r e c i p i t a t e d w i t h 0 . 1 v o l u m e s o d i u m a c e t a t e p H 5 . 4 a n d 2 . 2 5 v o l u m e s a b s o l u t e e t h a n o l i n a l i q u i d n i t r o g e n b a t h f o r 5 m i n . A f t e r c e n t r i f u g a t i o n f o r 30 m i n , t h e RNA p e l l e t was w a s h e d t w i c e w i t h 70% e t h a n o l a n d r e s u s p e n d e d i n s t e r i l e d e i o n i z e d d H 2 0 . The RNA was q u a n t i f i e d s p e c t r o p h o t o m e t r i c a l l y u s i n g a n e x t i n c t i o n c o e f f i c i e n t o f 25 (mg/ml) ~ 1 c m " 1 a t 260 nm. E l e c t r o p h o r e s i s o f d e n a t u r e d RNA was d o n e i n a 1% a g a r o s e g e l c o n t a i n i n g 5 mM m e t h y l m e r c u r i c h y d r o x i d e u s i n g a m o d i f i c a t i o n o f t h e m e t h o d o f B a i l e y a n d D a v i d s o n ( 1 ) . The g e l was r u n f o r 1 h r a t 100 V a n d s t a i n e d w i t h e t h i d i u m b r o m i d e (1 /^g/ml) a f t e r t h e a d d i t i o n o f 0.07% 2 - m e r c a p t o e t h a n o l . RNA s t a n d a r d s c o n s i s t e d o f a n RNA l a d d e r f r o m 9 . 5 t o 0 . 3 Kb ( B e t h e s d a R e s e a r c h L a b o r a t o r i e s ) . D o u b l e - s t r a n d e d RNA (dsRNA) was p u r i f i e d b y t h e m e t h o d o f K u r p p a a n d M a r t i n ( 4 6 ) . T w e n t y g r a m s a m p l e s o f i n f e c t e d C . q u i n o a t i s s u e w e r e p o w d e r e d i n l i q u i d n i t r o g e n a n d e x t r a c t e d w i t h 2 v o l u m e s o f 2x S T E ( l x = 50 mM T r i s , 100 mM N a C l a n d 1 mM E D T A pH 7 . 1 ) , 0 . 5 v o l u m e s 10% SDS a n d 1 . 5 v o l u m e s S T E s a t u r a t e d p h e n o l . The a q u e o u s p h a s e was a d j u s t e d t o 16% e t h a n o l a n d p o u r e d o v e r 2 . 5 g C F - 1 1 c e l l u l o s e (Whatman) , w a s h e d w i t h S T E c o n t a i n i n g 16% e t h a n o l ( S T E - E t O H ) a n d t h e b o u n d n u c l e i c a c i d s e l u t e d w i t h 15 m l S T E . S a m p l e s w e r e d i g e s t e d s e q u e n t i a l l y w i t h T 1 R N A a s e a n d D N A a s e 1 (Sigma) a d j u s t e d t o 20% E t O H a n d 1 /30 v o l u m e 0 . 5 M E D T A was a d d e d . T h e m a t e r i a l was b o u n d t o APX c e l l u l o s e ( S e r v a ) a n d w a s h e d o n a c o l u m n w i t h S T E - E t O H , t h e n e l u t e d w i t h S T E a n d p r e c i p i t a t e d w i t h 0 . 1 v o l u m e 3M s o d i u m a c e t a t e pH 5 . 2 a n d 2 . 5 v o l u m e s E t O H . S a m p l e s w e r e s t o r e d a t - 2 0 C . E l e c t r o p h o r e s i s o f t h e d s R N A was i n 1% a g a r o s e g e l s w i t h 1 / xg /ml e t h i d i u m b r o m i d e made w i t h T A E b u f f e r (40 mM T r i s , 40 mM a c e t a t e a n d 2 mM E D T A , pH 8 .0 ) f o r 2 -3 h r a t 4 0 V . S t a n d a r d s w e r e P s t I o r H i n d I I I c u t l a m b d a p h a g e D N A . To d e t e r m i n e b u o y a n t d e n s i t y a s m a l l amount o f c a e s i u m s u l p h a t e was w i t h d r a w n f r o m a s i s t e r t u b e a t t h e same l e v e l as t h e v i r u s b a n d a f t e r i s o p y c n i c c e n t r i f u g a t i o n a n d t h e r e f r a c t i v e i n d e x r e a d on a r e f r a c t o m e t e r a n d c o n v e r t e d t o d e n s i t y u s i n g t h e f o r m u l a o f V i n o g r a d a n d H e a r s t ( 7 8 ) . 37 R e s u l t s T h e p a r t i c l e s w e r e d i s r u p t e d i n 2% P T A a n d o n l y 2% UA was u s e d . P u r i f i e d v i r u s p a r t i c l e s w e r e l o n g , v e r y f l e x u o u s r o d s w i t h a s t r i a t e d c r o s s - b a n d i n g p a t t e r n . The a x i a l c a n a l was n o t v i s i b l e a n d f u l l l e n g t h a n d f r a g m e n t e d p a r t i c l e s w e r e o b s e r v e d , f r e q u e n t l y i n t e r t w i n e d i n a g g r e g a t e s w i t h h o s t m a t e r i a l . A l s o , s m a l l (10 -12 nm) i r r e g u l a r s p h e r e s w e r e f o u n d ( F i g . 2 ) . M e a s u r e m e n t s o f p a r t i c l e w i d t h f o r t h e B i s o l a t e made f r o m f i v e p a r t i c l e s f r o m f i v e s e p a r a t e p u r i f i c a t i o n s a v e r a g e d 1 1 . 5 nm a n d t h e h e l i c a l p i t c h m e a s u r e d f r o m t h r e e p a r t i c l e s f r o m t h r e e s e p a r a t e p u r i f i c a t i o n s a v e r a g e d 3 . 2 5 nm. T h e p a r t i c l e l e n g t h d i s t r i b u t i o n f o r t h e A i s o l a t e was p l o t t e d u s i n g a h i s t o g r a m c e l l w i d t h o f 50 nm ( F i g . 4 ) . T h e m o d a l l e n g t h was 782 nm a n d t h e t h r e e c o n t i g u o u s c e l l s a r o u n d t h e mode c o n t a i n e d 71% o f t h e p a r t i c l e s m e a s u r e d . T h e l e n g t h d i s t r i b u t i o n o f t h e B i s o l a t e ( F i g . 5) was more d i f f i c u l t t o i n t e r p r e t . The l e n g t h m o s t f r e q u e n t l y o b s e r v e d i s 600 t o 650 nm a n d w i t h t h e two c o n t i g u o u s c e l l s i n c l u d e s 39.5% o f t h e p a r t i c l e s , h o w e v e r , a n o t h e r p e a k o c c u r s a r o u n d t h e 750 t o 800 nm c e l l w h i c h a l s o c o n t a i n s 39.5% o f t h e p a r t i c l e s . The m a j o r i t y o f p a r t i c l e s f e l l i n t h e 550 t o 900 nm r a n g e , t h e a v e r a g e o f t h i s g r o u p b e i n g 732 nm. T h i n s e c t i o n s f r o m b o t h P . g l a n d u l o s a i s o l a t e s a n d i n f e c t e d C . q u i n o a w e r e e x a m i n e d f o r e v i d e n c e o f p a r t i c l e s i n v i v o a n d f o r c y t o p l a s m i c i n c l u s i o n s s u c h as v e s i c l e s o r c r o s s - b a n d e d b o d i e s w h i c h h a v e b e e n a s s o c i a t e d w i t h o t h e r c l o s t e r o v i r u s e s ( 2 5 ) . I n 38 1 2 10 -8 -425 i i i i r 525 625 725 825 925 P a r t i c l e L e n g t h (nm) n i r 1025 F i g . 4. Modal length d i s t r i b u t i o n of 46 p a r t i c l e s of ACLSV i s o l a t e A measured from p u r i f i e d p r e p a r a t i o n s . Histogram c e l l width = 50 nm. Modal length = 782 nm. 425 525 625 725 825 925 1025 P a r t i c l e L e n g t h (nm) F i g . 5 . M o d a l l e n g t h d i s t r i b u t i o n o f 38 p a r t i c l e s o f A C L S V i s o l a t e B m e a s u r e d f r o m p u r i f i e d p r e p a r a t i o n s . H i s t o g r a m c e l l w i d t h = 50 nm. M o d a l l e n g t h = 732 nm. 40 t h e woody h o s t s , l o n g f l e x u o u s s t r a n d s w e r e o b s e r v e d i n s i e v e t u b e members a n d c o m p a n i o n c e l l s b u t n o t i n t h e m e s o p h y l l . The s t r a n d s w e r e f o u n d l o o s e w i t h i n t h e c e l l o r a s s o c i a t e d w i t h s i e v e p l a t e s ( F i g . 6 A ) . I n C . q u i n o a t h i n f l e x u o u s r o d s w e r e s e e n l o o s e l y a r r a n g e d i n y o u n g s i e v e - t u b e members i n t h e c y t o p l a s m a n d n u c l e u s a n d a l s o a l i g n e d i n p a r a l l e l b u n d l e s ( F i g . 6 B ) . Some b u n d l e s a p p e a r e d i n c r o s s - s e c t i o n as a t i g h t l y p a c k e d a r r a y o f d o t s . No v e s i c l e s w e r e f o u n d . I t i s n o t known i f t h e f l e x u o u s r o d s w e r e P - p r o t e i n , w h i c h i s f o u n d i n t h e s i e v e - t u b e members o f d i c o t y l e d o n o u s p l a n t s , o r v i r i o n s . T h r e e v i r u s s p e c i f i c dsRNA b a n d s w e r e e x t r a c t e d f r o m A C L S V i n f e c t e d C . q u i n o a o f MW 5 . 6 x 1 0 6 , 4 . 8 8 x 1 0 6 a n d 4 . 4 5 x 1 0 6 Da ( F i g . 7 ) . T h e s s R N A e x t r a c t e d f r o m p u r i f i e d i s o l a t e A h a d a m a j o r b a n d a t 2 . 6 6 x 1 0 6 a n d m i n o r b a n d s a t 1 . 5 3 x 1 0 6 a n d 0 . 6 5 x 1 0 6 Da ( F i g . 8 ) . T h e b u o y a n t d e n s i t y o f A C L S V i s o l a t e s A a n d B i n c a e s i u m s u l p h a t e was 1 . 2 6 g / c c 3 a n d 1 . 2 8 g / c c 3 r e s p e c t i v e l y . The c o a t p r o t e i n MW o f t h e A C L S V i s o l a t e s was e s t i m a t e d a t 2 4 . 5 KDa (B) a n d 2 6 . 0 KDa (A) ( T a b l e 3 ) . The s l i g h t d i f f e r e n c e i n MW b e t w e e n t h e two i s o l a t e s was c o n s i s t e n t o v e r f o u r d i f f e r e n t g e l s . T h e r e w e r e a l s o m a j o r h o s t c o m p o n e n t c o n t a m i n a t i n g b a n d s a t 49 a n d 66 K D a . T h a t t h e s e b a n d s w e r e n o t o f v i r a l o r i g i n was c o n f i r m e d b y W e s t e r n b l o t a n a l y s i s w h e r e t h e membrane was s o a k e d i n a n t i b o d y d i l u t e d i n c l a r i f i e d h e a l t h y C . q u i n o a s a p a n d p r o b e d w i t h 1 2 5 I - l a b e l l e d P r o t e i n - A . T h e 2 4 . 5 a n d 26 KDa b a n d s s h o w e d s t r o n g r e a c t i o n s w h i l e t h e r e was o n l y a f a i n t r e a c t i o n a t t h e 49 KDa b a n d w i t h t h e more c o n t a m i n a t e d B i s o l a t e ( F i g . 9 ) . F i g . 6. V i r u s - l i k e p a r t i c l e s i n a p p l e c h l o r o t i c l e a f s p o t v i r u s i n f e c t e d t i s s u e s . A . P a r t i c l e s a s s o c i a t e d w i t h a s i e v e - p l a t e i n i n f e c t e d P r u n u s g l a n d u l o s a . B a r = 1/zm. B . B u n d l e s o f v i r u s - l i k e p a r t i c l e s i n a s i e v e - t u b e member o f i n f e c t e d C h e n o p o d i u m q u i n o a B a r = 1/um. A r r o w s = b u n d l e s i n c y t o p l a s m a n d n u c l e u s . 42 F i g . 7 . E l e c t r o p h o r e t i c v i s u a l i z a t i o n o f d s R N A e x t r a c t e d f r o m a p p l e c h l o r o t i c l e a f s p o t i n f e c t e d C h e n o p o d i u m q u i n o a . L a n e A = P s t I c u t l a m b d a , L a n e B = i s o l a t e A , L a n e C = i s o l a t e B , L a n e D = h e a l t h y C . q u i n o a , L a n e E = H i n d l l l c u t l a m b d a . L i n e m a r k s p o s i t i o n o f t h r e e v i r u s - s p e c i f i c b a n d s a t MW 5 . 6 x 1 0 6 , 4 . 8 8 x 1 0 6 a n d 4 . 4 5 x 1 0 6 . 43 A B C D 7.7Kb- C23 — -» 1 mm m F i g . 8. E l e c t r o p h o r e t i c v i s u a l i z a t i o n o f s s R N A e x t r a c t e d f r o m a p p l e c h l o r o t i c l e a f s p o t i n f e c t e d C h e n o p o d i u m q u i n o a . L a n e s A & D = RNA l a d d e r f r o m 9 . 5 t o 0 . 3 K b , L a n e s B & C = i s o l a t e A . 44 T a b l e 3 . P h y s i c a l c h a r a c t e r i s t i c s o f A C L S V f r o m P . g l a n d u l o s a C o a t P r o t e i n (kDa) SSRNA (x 10 6 ) d s R N A (x 10 6 ) A C L S V - A 2 6 . 0 2 . 66 5 . 6 0 1 . 5 3 4 . 8 8 0 . 6 5 4 .45 A C L S V - B 24 .5 - 5 . 60 4 . 8 8 4 .45 45 F i g . 9 . A . E l e c t r o p h o r e t i c v i s u a l i z a t i o n a n d W e s t e r n B l o t o f a p p l e c h l o r o t i c l e a f s p o t v i r u s c o a t p r o t e i n . S t d = l o w m o l e c u l a r w e i g h t s t a n d a r d s ( 9 2 . 5 t o 1 4 . 4 K D a ) , 1114 = i s o l a t e A , C . q . s a p = raw C h e n o p o d i u m q u i n o a s a p , C l a r s a p = c l a r i f i e d C . q u i n o a s a p , 1292 = i s o l a t e B . V i r u s c o a t p r o t e i n b a n d = 24 t o 26 K D a . B . W e s t e r n b l o t o f F i g . 9A g e l p r o b e d w i t h i s o l a t e B p o l y c l o n a l . 125 a n t i s e r u m d i l u t e d w i t h c l a r i f i e d C . q u i n o a s a p f o l l o w e d b y I -l a b e l l e d P r o t e i n A . 46 D i s c u s s i o n C l o s t e r o v i r u s e s e x h i b i t some u n i q u e c y t o p a t h o l o g i c a l p r o p e r -t i e s w h i c h c a n b e d i a g n o s t i c t o t h e g r o u p l e v e l (35) a n d o t h e r s w h i c h a r e u s e d t o d i v i d e t h e m i n t o s u b g r o u p s ( 2 5 ) . B a n d e d b o d i e s i n t h e p h l o e m a r e c h a r a c t e r i s t i c o f c l o s t e r o v i r u s e s w h i l e w i t h i n t h e g r o u p , B Y V - t y p e v e s i c l e s a r e i n d i c a t i v e o f s u b g r o u p I ( s u b g r o u p B a c c o r d i n g t o B a r - J o s e p h a n d M u r a n t , ( 5 ) ) . A C L S V i s p l a c e d i n s u b g r o u p I I . A common p r o b l e m i n i n t e r p r e t i n g t h e c y t o p a t h o l o g i c a l e f f e c t s o f c l o s t e r o v i r u s e s i s d i s t i n g u i s h i n g t h e m f r o m P - p r o t e i n w h i c h may o c c u r i n s e v e r a l f o r m s ( g r a n u l a r , f i l a m e n t o u s , f i b r i l l a r , t u b u l a r a n d c r y s t a l l i n e ) i n t h e s i e v e - t u b e members o f d i c o t y l e d o n o u s p l a n t s a n d i n some m o n o c o t s . T h e f u n c t i o n o f P -p r o t e i n i s u n k n o w n b u t p o s s i b l e r o l e s i n c l u d e t h e s e a l i n g o f s i e v e - p l a t e p o r e s i n i n j u r e d s i e v e t u b e s o r l o n g d i s t a n c e t r a n s p o r t o f s u b s t a n c e s i n t h e p h l o e m ( 2 3 ) . O c c a s i o n a l l y , P -p r o t e i n a n d v i r i o n s c a n b e d i s t i n g u i s h e d i n t h e same m i c r o g r a p h b u t i n t h i s w o r k i t i s n o t known i f t h e f i l a m e n t o u s m a t e r i a l o b s e r v e d i n t h e m i c r o g r a p h s i s P - p r o t e i n o r v i r u s p a r t i c l e s . I m m u n o g o l d s t a i n i n g w o u l d b e a u s e f u l t e c h n i q u e t o make t h i s d i s t i n c t i o n , h o w e v e r an a n t i s e r u m w i t h o u t a n y b a c k g r o u n d r e a c -t i v i t y a g a i n s t h o s t c o m p o n e n t s w o u l d b e n e e d e d . I n c o n t r a s t t o t h e r e s u l t s o f O h k i e t a l . (61) no e v i d e n c e o f A C L S V v i r u s p a r t i c l e s was f o u n d o u t s i d e o f t h e v a s c u l a r t i s s u e . T h i s may b e b e c a u s e d i f f e r e n t v i r u s i s o l a t e s w e r e s t u d i e d . T h e p a r t i c l e m o d a l l e n g t h s f o r t h e A a n d B i s o l a t e s (782 a n d 732 nm r e s p e c t i v e l y ) a r e c l o s e t o t h e p r e v i o u s l y r e p o r t e d v a l u e s o f 7 95 nm f o r a n A C L S V i s o l a t e f r o m P r u n u s d o m e s t i c a . T h e p a r t i c l e m e a s u r e m e n t s w e r e 30 nm l o n g e r (825 v s 795) when m e a s u r e d f r o m l e a f s a p as c o m p a r e d t o p u r i f i e d v i r u s ( 7 2 ) . V a l u e s f o r a p p l e i s o l a t e s o f A C L S V a r e b e t w e e n 620 t o 740 nm. T h e d i f f e r e n c e i n m o d a l l e n g t h s b e t w e e n t h e A a n d B i s o l a t e s may b e r e a l o r s i m p l y r e f l e c t t h e i n c r e a s e d s u s c e p t i b i l i t y t o b r e a k a g e b y t h e B i s o l a t e d u r i n g p u r i f i c a t i o n . By r e p o r t i n g t h e m o d a l l e n g t h o f r o d - s h a p e d v i r u s e s , t h e e f f e c t s o f e n d t o e n d a g g r e g a t i o n c a n b e e l i m i n a t e d a n d v i r u s e s w i t h p a r t i c l e s o f two s i z e s ( e g . t o b a c c o r a t t l e ) c a n b e d e t e c t e d . H o w e v e r , when e a s i l y d a m a g e d r o d s f r o m p u r i f i e d p r e p a r a t i o n s a r e u s e d f o r m o d a l l e n g t h d e t e r m i n a t i o n , t h e v a l u e r e p o r t e d may b e t h e l e n g t h t o w h i c h t h e p a r t i c l e s a r e m o s t f r e q u e n t l y b r o k e n a n d n o t t h e t r u e l e n g t h o f t h e v i r u s i n v i v o . A t t e m p t s t o a n s w e r t h i s q u e s t i o n b y t r a p p i n g p a r t i c l e s f r o m c l a r i f i e d s a p w e r e n o t s u c c e s s f u l , p r o b a b l y b e c a u s e t h e p o l y c l o n a l a n t i s e r u m h a d a l a r g e c o m p o n e n t d i r e c t e d a g a i n s t h o s t p r o t e i n s o r t h e v i r u s c o n c e n t r a t i o n was t o o l o w . T h e p a r t i c l e w i d t h o f 1 1 . 5 nm i s c o n s i s t e n t w i t h o t h e r e s t i m a t e s f o r A C L S V o f 12 nm (4) as i s t h e p i t c h e s t i m a t e o f 3 . 2 5 nm c o m p a r e d w i t h a r e p o r t e d v a l u e o f 3 . 8 nm ( 3 ) . T h e d i f f e r e n c e i n c o a t p r o t e i n MW b e t w e e n A C L S V i s o l a t e s A a n d B i s s m a l l a n d b o t h e s t i m a t e s a r e w i t h i n t h e r a n g e r e p o r t e d p r e v i o u s l y f o r A C L S V ( 8 1 , 9 ) . The f u n c t i o n o f v i r a l c o a t p r o t e i n s g o e s b e y o n d t h a t o f p r o t e c t i n g t h e v i r a l R N A . Dawson e t a l . (15) s h o w e d t h a t TMV m u t a n t s w i t h i n s e r t i o n s o r d e l e t i o n s i n t h e c o a t 48 p r o t e i n g e n e a f f e c t e d t h e t y p e o f symptoms e x p r e s s e d i n X a n t h i t o b a c c o . F u r t h e r r e s e a r c h s h o u l d b e c o n d u c t e d o n w h e t h e r t h e d i f f e r e n c e i n t h e c o a t p r o t e i n o f i s o l a t e A a n d B i s r e s p o n s i b l e f o r t h e l a c k o f s y s t e m i c movement i n C . a m a r a n t i c o l o r b y i s o l a t e A . T h e W e s t e r n b l o t a n a l y s i s c o n f i r m e d t h a t t h e 2 4 . 5 a n d 26 KDa p r o t e i n s w e r e o f v i r a l o r i g i n . A f t e r c r o s s - a d s o r p t i o n o f t h e d e t e c t i n g a n t i b o d y w i t h c l a r i f i e d C . q u i n o a s a p , t h e f a i n t r e a c t i o n w i t h t h e 49 KDa b a n d i n d i c a t e s t h a t t h e p o l y c l o n a l a n t i s e r u m c o n t a i n s a c o m p o n e n t d i r e c t e d a g a i n s t t h i s h o s t p r o t e i n w h i c h c o u l d n o t b e r e m o v e d . D o u b l e - s t r a n d e d RNA a n a l y s i s h a s b e e n u s e d as a d i a g n o s t i c t o o l f o r d e t e c t i o n a n d c l a s s i f i c a t i o n o f c l o s t e r o v i r u s e s . D o d d s a n d B a r - J o s e p h (19) r e p o r t e d a dsRNA o f MW 4 . 6 X 1 0 6 a n d t h r e e m i n o r b a n d s a t a p p r o x i m a t e l y 1 . 5 X 1 0 6 . I n t e r e s t i n g l y , t h i s p a t t e r n was n o t s i m i l a r t o t h e t h r e e o t h e r c l o s t e r o v i r u s e s a n d t h e a u t h o r s q u e s t i o n e d t h e i n c l u s i o n o f A C L S V i n t h e c l o s t e r o v i r u s g r o u p . Y o s h i k a w a a n d T a k a h a s h i (81) f o u n d t h r e e m a j o r (MW (X 10 6 ) 4 . 9 , 4 . 3 , 3 .9 ) a n d two m i n o r ( 4 . 5 a n d 3 .1 ) b a n d s f r o m A C L S V i n f e c t e d C . q u i n o a . B o t h A a n d B i s o l a t e s s h o w e d s i m i l a r d s R N A p a t t e r n s c o n s i s t i n g o f t h r e e b a n d s (MW (X 10 6 ) 5 . 6 0 , 4 . 8 8 , 4 . 4 5 ) i n t h e same r e l a t i v e p o s i t i o n a s t h e t h r e e b a n d s r e p o r t e d b y Y o s h i k a w a a n d T a k a h a s h i (81) b u t a t h i g h e r m o l e c u l a r w e i g h t s . I t i s n o t known i f t h i s i s d u e t o t h e s o u r c e o f t h e i s o l a t e s b e i n g P r u n u s r a t h e r t h a n M a l u s o r r e f l e c t s t h e d i f f e r e n t t e c h n i q u e s u s e d . The MW o f t h e s l o w e s t m i g r a t i n g b a n d ( 5 . 6 6 X 10 ) was s l i g h t l y more t h a n d o u b l e t h a t o f t h e m a j o r 49 ssRNA band (2.66 X 10 6) . With the e x c e p t i o n of the v i r i o n s not b e i n g found i n the mesophyll, the p h y s i c a l p r o p e r t i e s and cytopat h o l o g y of the A and B i s o l a t e s agree with those p r e v i o u s l y r e p o r t e d f o r ACLSV. 50 C h a p t e r 5 - S e r o l o g i c a l D e t e c t i o n I n t r o d u c t i o n A C L S V h a s b e e n d e t e c t e d b y s e r o l o g i c a l t e c h n i q u e s s u c h a s r i n g i n t e r f a c e ( 6 7 ) , a g a r g e l d i f f u s i o n ( 9 ) , E L I S A ( 2 4 , 1 8 , 2 7 , 2 , 8 0 , 6 6 ) , a n d i m m u n o s o r b e n t e l e c t r o n m i c r o s c o p y (ISEM) ( 4 2 , 4 4 ) . S a k s e n a a n d M i n k (67) c o u l d d e t e c t A C L S V f r o m b l o s s o m s b u t n o t f r o m l e a f t i s s u e b y t h e r i n g p r e c i p i t i n t e s t . T h e v i r u s f r o m b l o s s o m s r e q u i r e d a p a r t i a l p u r i f i c a t i o n b e f o r e b e i n g u s e d . A g a r g e l d i f f u s i o n t e s t s c a n b e d o n e w i t h i n f e c t e d C . q u i n o a s a p b u t n o t f r o m M a l u s o r P r u n u s l e a f t i s s u e ( 9 ) . T h e o m i s s i o n o f s t a b i l i z i n g a g e n t s s u c h a s p o l y a m i n e s o r M g C l 2 d e g r a d e s t h e v i r u s a n d i m p r o v e s d i f f u s i o n t h r o u g h t h e a g a r ( 5 2 ) . B e c a u s e o f t h e d i f f i c u l t i e s i n d e t e c t i n g A C L S V i n woody h o s t s b y g e l d i f f u s i o n a n d r i n g p r e c i p i t i n t e s t s , t h e E L I S A t e c h n i q u e i s p r e f e r r e d f o r r o u t i n e d e t e c t i o n . F l e g g a n d C l a r k (24) u s e d a m o d i f i c a t i o n o f t h e b a s i c t e c h n i q u e w h e r e t h e s a m p l e was i n c u b a t e d s i m u l t a n e o u s l y w i t h t h e a n t i b o d y - c o n j u g a t e . T h e y s p e c u l a t e d t h a t t h e v i r u s i n t h e v i r u s - a n t i b o d y c o m p l e x e s f o r m e d b y t h i s s t e p w e r e l e s s s u s c e p t i b l e t o d e g r a d a t i o n . D e t i e n n e e t a l (18) f o u n d t h a t t h e s t a n d a r d E L I S A m e t h o d c o u l d b e u s e d i f M g C l 2 , 3 , 3 ' -d i a m i n o d i p r o p y l a m i n e (DD) a n d n i c o t i n e w e r e a d d e d t o t h e e x t r a c t i o n b u f f e r a n d t h i s m e t h o d c o u l d d e t e c t A C L S V f r o m a l l s o u r c e s e x c e p t a p r i c o t . A F ( a b ' ) 2 b a s e d E L I S A was u s e d b y B a r b a a n d C l a r k (2) t o d e t e c t A C L S V i n a l l woody h o s t s e x c e p t p e a r . M o n o c l o n a l a n t i b o d y t e c h n o l o g y , f i r s t d e v e l o p e d b y K o h l e r a n d M i l s t e i n ( 4 5 ) , h a s a l l o w e d f o r t h e r o u t i n e u s e o f s e r o l o g i c a l 51 techniques without the problems a s s o c i a t e d w i t h p o l y c l o n a l antiserum such as l i m i t e d supply, v a r i a b l e q u a l i t y and non-s p e c i f i c r e a c t i o n s (57). Host components are o f t e n found i n p u r i f i e d ACLSV preparations and p o l y c l o n a l antiserum produced from these preparations must be cross-adsorbed w i t h host antigens. To avoid t h i s problem Poul and Dunez (66) produced monoclonal a n t i b o d i e s against the plum bark s p l i t s t r a i n of ACLSV and developed an ELISA t e s t with t e n - f o l d increased s e n s i t i v i t y over a t e s t using p o l y c l o n a l antiserum. The s e r o l o g i c a l v a r i a b i l i t y of ACLSV was f i r s t reported by Chairez and L i s t e r (9) when they found d i f f e r e n c e s between s t r a i n s from Malus and Prunus. In co n t r a s t Detienne et a l . (18) found l i m i t e d a n t i g e n i c v a r i a t i o n among ACLSV s t r a i n s using p o l y c l o n a l antiserum against a Prunus s t r a i n . Barba and Cl a r k (2) showed th a t antiserum prepared against an apple i s o l a t e reacted w e l l w i t h homologous antigens but d i d not detect a Prunus i s o l a t e causing v i r u e l a of a p r i c o t . Conversely, an antiserum prepared against a plum i s o l a t e detected i s o l a t e s from Prunus and apple. These r e p o r t s of s e r o l o g i c a l v a r i a t i o n emphasize the need f o r adequate screening of the antiserum s p e c i f i c i t y before adopting a s e r o l o g i c a l t e s t i n g program. For example, a commercially a v a i l a b l e ACLSV antiserum f a i l e d t o detect the v i r u s i s o l a t e d from apple t r e e s i n F i n l a n d (50). M a t e r i a l s and Methods P o l y c l o n a l antiserum against the A and B ACLSV i s o l a t e s , and 52 h e a l t h y C . q u i n o a w e r e p r o d u c e d i n New Z e a l a n d W h i t e r a b b i t s . The i m m u n i z a t i o n s c h e d u l e c o n s i s t e d o f f o u r w e e k l y i n t r a m u s c u l a r i n j e c t i o n s o f 0 . 1 t o 0 . 5 mg a n t i g e n w i t h F r e u n d ' s i n c o m p l e t e a d j u v a n t f o l l o w e d a week l a t e r b y one i n t e r v e i n a l i n j e c t i o n o f 0 . 2 t o 0 . 3 mg w i t h o u t a d j u v a n t . T e n a n d 15 d a y s l a t e r t h e r a b b i t s w e r e b l e d . G a m m a - g l o b u l i n s w e r e p u r i f i e d b y ammonium s u l p h a t e p r e c i p i t a t i o n a n d p a s s a g e t h r o u g h DEAE c e l l u l o s e , t h e n a d j u s t e d t o 1 m g / m l u s i n g an A 2 8 0 = 1 . 4 . C o n j u g a t i o n o f a n t i b o d i e s w i t h a l k a l i n e p h o s p h a t a s e was a c c o r d i n g t o C l a r k a n d Adams (10) u s i n g g l u t a r a l d e h y d e . T h e d o u b l e a n t i b o d y s a n d w i c h (DAS) E L I S A was d o n e a c c o r d i n g t o C l a r k a n d Adams (10) w i t h a f e w m o d i f i c a t i o n s . L i n b r o p o l y s t y r e n e p l a t e s ( F l o w L a b o r a t o r i e s ) w e r e c o a t e d w i t h a n t i b o d y i n c o a t i n g b u f f e r f o r 1 h r a t 37 C f o l l o w e d b y p h o s p h a t e b u f f e r e d s a l i n e (PBS) w i t h 0.05% T w e e n - 2 0 a n d 0.1% B l o t t o (PTB) f o r 1 h r a t r o o m t e m p e r a t u r e . S a m p l e s g r o u n d 1 : 1 0 (w/v) i n PTB (2% PVP a d d e d f o r woody t i s s u e ) w e r e a d d e d t o t h e w e l l s a n d l e f t o v e r n i g h t a t 4 C . T h e s p e c i f i c c o n j u g a t e s w e r e d i l u t e d i n h e a l t h y C . q u i n o a s a p , e i t h e r f r e s h o r p r e v i o u s l y f r o z e n , g r o u n d 1 : 1 0 (w/v) w i t h P T B a n d f i l t e r e d t h r o u g h two l a y e r s o f K i m w i p e s , l e f t 10 m i n a n d t h e n a d d e d t o t h e p l a t e s f o r 3 h r a t 37 C . F o r c o m p a r i s o n , t h e a p p r o p r i a t e amount o f s t o c k c o n j u g a t e s w e r e a d d e d t o a v o l u m e o f h e a l t h y C . q u i n o a e x t r a c t ( 1 : 1 0 w / v ) e q u a l t o 1 /10 t h e v o l u m e o f d i l u t e d c o n j u g a t e r e q u i r e d f o r t h e t e s t , l e f t 10 m i n a t r o o m t e m p e r a t u r e (RT) a n d t h e n d i l u t e d w i t h P T B a n d a d d e d t o t h e p l a t e s ( 3 3 ) . A f t e r t h e a d d i t i o n o f s u b s t r a t e t a b l e t s , ( p -n i t r o p h e n y l p h o s p h a t e ) (Sigma) t h e p l a t e s w e r e r e a d a t 405 nm on 53 an a u t o m a t e d p l a t e r e a d e r ( T i t e r t e k F l o w L a b o r a t o r i e s ) a f t e r 1 t o 2 h r a n d a f t e r s t a n d i n g o v e r n i g h t . I n a t t e m p t s t o l o w e r b a c k g r o u n d r e a c t i o n s , A C L S V p o l y c l o n a l a n t i s e r u m was c r o s s a d s o r b e d w i t h h o s t p r e p a r a t i o n s . H e a l t h y C . q u i n o a was p u r i f i e d t o t h e PEG p r e c i p i t a t i o n s t a g e d e s c r i b e d i n t h e v i r u s p u r i f i c a t i o n c h a p t e r . One h a l f a m i l l i l i t r e o f t h i s p r e p a r a t i o n was a d d e d t o 1 m l o f a n t i s e r u m a n d 8 . 5 m l o f d H 2 0 , l e f t 16 h r a t RT a n d c e n t r i f u g e d 20 m i n a t 1 4 , 0 0 0 g f o l l o w e d b y ammonium s u l p h a t e p r e c i p i t a t i o n a n d p u r i f i c a t i o n o f i m m u n o g l o b u l i n s on DEAE c e l l u l o s e . A g a r g e l d o u b l e d i f f u s i o n t e s t s w e r e made i n 1.0% a g a r g e l s c o n t a i n i n g 0.5% s o d i u m d o d e c y l s u l p h a t e ( S D S ) , 0.85% N a C l a n d 0.02% s o d i u m a z i d e . T h r e e h u n d r e d m i c r o l i t r e s o f 10% SDS w e r e a d d e d t o t h e TM g r i n d i n g b u f f e r f o r e a c h g r a m o f s a m p l e . M o n o c l o n a l a n t i b o d i e s (MABs) a g a i n s t t h e A i s o l a t e w e r e p r o d u c e d b y i n j e c t i n g B a l b / c m i c e s u b c u t a n e o u s l y w i t h 35 fj.g o f p u r i f i e d v i r u s d i s r u p t e d w i t h 1% SDS a n d m i x e d e q u a l l y w i t h F r e u n d ' s i n c o m p l e t e a d j u v a n t . A f t e r a 25 d a y r e s t t h e m i c e were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 50 fj.g v i r u s a n d s a c r i f i c e d 4 d a y s l a t e r t o r e m o v e t h e i r s p l e e n . F o x - N Y m y e l o m a c e l l s w e r e c u l t u r e d i n D u l b e c c o ' s M o d i f i e d E a g l e ' s m e d i a (DME) p l u s 10% f e t a l c a l f s e r u m (FCS) ( H y c l o n e , L o g a n , U t a h ) a n d f u s e d w i t h s p l e n o c y t e s a c c o r d i n g t o t h e m e t h o d o f K o h l e r a n d M i l s t e i n (45) u s i n g 50% P E G . T h e c e l l s w e r e r e s u s p e n d e d i n 20% FCS DME w i t h a m i n o p t e r i n , h y p o x t h a n i n e , a d e n i n e , t h y m i n e a n d a t h y m o c y t e f e e d e r l a y e r . A n t i b o d y s e c r e t i n g h y b r i d o m a s w e r e i d e n t i f i e d b y a n i n d i r e c t t r i p l e a n t i b o d y s a n d w i c h (TAS) E L I S A . H a l f o f t h e p l a t e s w e r e c o a t e d w i t h i s o l a t e A p o l y c l o n a l a n t i b o d i e s a t 2 /j.g/m.1 f o r 1 . 5 h r a t 37 C f o l l o w e d b y a b l o c k i n g s t e p w i t h P T B . H e a l t h y C . q u i n o a s a p g r o u n d 1 : 1 0 i n PTB was l e f t on t h e p l a t e s f o r 16 h r . C u l t u r e f l u i d (90 ptl) was t h e n a d d e d f o r 1 h r a t 37 C , f o l l o w e d b y a r a b b i t a n t i - m o u s e a l k a l i n e p h o s p h a t a s e c o n j u g a t e ( J a c k s o n Immuno R e s e a r c h ) f o r 1 h r a t 37 C a n d t h e n P - n i t r o p h e n y l p h o s p h a t e t a b l e t s i n s u b s t r a t e b u f f e r . T h e o t h e r h a l f o f t h e p l a t e s w e r e d i r e c t c o a t e d w i t h p u r i f i e d i s o l a t e A v i r u s ( 2 . 5 /ug/ml) i n c o a t i n g b u f f e r f o r 16 h r a t R T , b l o c k e d w i t h P T B , a n d f o l l o w e d b y t h e p r o c e d u r e s d e s c r i b e d a b o v e . C l o n e s w h i c h r e a c t e d t o d i r e c t c o a t e d v i r u s a n d n o t h o s t a n t i g e n s w e r e s e l e c t e d f o r f u r t h e r t e s t i n g . P o s i t i v e c e l l l i n e s w e r e c l o n e d b y l i m i t i n g d i l u t i o n u s i n g a t h y m o c y t e f e e d e r l a y e r , g r o w n i n c e l l c u l t u r e a n d e i t h e r i n j e c t e d i n t r a p e r i t o n e a l l y i n t o m i c e f o r p r o d u c t i o n o f a s c i t i c f l u i d (62) o r g r o w n i n 1 L c u l t u r e b o t t l e s . A n t i b o d i e s w e r e p u r i f i e d f r o m a s c i t e s f l u i d b y p r e c i p i t a t i o n w i t h 50% s a t u r a t e d ammonium s u l p h a t e f o l l o w e d b y p a s s a g e t h r o u g h a D E A E c e l l u l o s e c o l u m n . One l i t r e b a t c h c u l t u r e s w e r e p u r i f i e d b y l o w s p e e d c e n t r i f u g a t i o n t o r e m o v e h y b r i d o m a c e l l s a n d f o l l o w e d b y f r e e z e d r y i n g o r p r e c i p i t a t i o n w i t h ammonium s u l p h a t e . T h e s u b c l a s s o f e a c h a n t i b o d y was d e t e r m i n e d w i t h a h y b r i d o m a i m m u n o g l o b u l i n t y p i n g k i t ( B e h r i n g D i a g n o s t i c s ) . T h e t r i p l e a n t i b o d y s a n d w i c h (TAS) E L I S A was u s e d t o t e s t t h e MABs a g a i n s t t h e A C L S V i s o l a t e s . One MAB was d i r e c t l y c o n j u g a t e d t o a l k a l i n e p h o s p h a t a s e a n d u s e d i n t h e D A S - E L I S A w i t h p o l y c l o n a l 55 c o a t i n g a n t i b o d y . A c o m p a r i s o n was made o f t h e f o l l o w i n g g r i n d i n g b u f f e r a d d i t i v e s i n t h e T A S - E L I S A s y s t e m : 2.5% n i c o t i n e , 0 . 0 1 M M g S 0 4 , 0.2% D D , n i c o t i n e p l u s M g S 0 4 p l u s D D , a n d n i c o t i n e p l u s M g S 0 4 . R e s u l t s T h e two p o l y c l o n a l A C L S V a n t i s e r a g a v e h i g h b a c k g r o u n d r e a c t i o n s w i t h o u t some f o r m o f c r o s s - a d s o r p t i o n a g a i n s t h e a l t h y C . q u i n o a ( T a b l e 4 ) . The s p e c i f i c a n t i b o d y - e n z y m e c o n j u g a t e f r o m b o t h t r e a t m e n t s was a l s o d i l u t e d i n h e a l t h y C . q u i n o a s a p o r P T B . T h e h i g h e s t i n f e c t e d / h e a l t h y r a t i o was a c h i e v e d w i t h u n a d s o r b e d a n t i s e r u m w h e r e t h e c o n j u g a t e was d i l u t e d i n h o s t s a p . A s e p a r a t e t e s t was d o n e c o m p a r i n g d i l u t i o n o f c o n j u g a t e i n s a p w i t h t h e m e t h o d o f G o n s a l v e s e t a l . (33) w h e r e t h e a p p r o p r i a t e v o l u m e o f s t o c k c o n j u g a t e was i n c u b a t e d w i t h a s m a l l v o l u m e o f h o s t s a p a n d t h e n d i l u t e d w i t h P T B . R e a c t i o n s t o h e a l t h y C . q u i n o a w e r e 1 . 6 t i m e s h i g h e r a n d r e a c t i o n s t o i n f e c t e d t i s s u e w e r e one h a l f t o one q u a r t e r l o w e r w i t h t h e l a t t e r m e t h o d . T h e d e t e c t i o n l i m i t o f h o m o l o g o u s p u r i f i e d v i r u s b y D A S - E L I S A was a p p r o x i m a t e l y 25 n g / m l . A C L S V was a l s o d e t e c t e d i n P . g l a n d u l o s a l e a v e s a n d f l o w e r s b u t was e r r a t i c a l l y d i s t r i b u t e d a n d t h e s i g n a l weak, t y p i c a l l y i n t h e r a n g e o f two t o f i v e t i m e s t h a t o f h e a l t h y C . q u i n o a . I n a g a r g e l d i f f u s i o n t e s t s w i t h p u r i f i e d v i r u s t h e A a n d B i s o l a t e s f o r m e d c o n t i n u o u s p r e c i p i t i n l i n e s ( F i g . 10) h o w e v e r t h i s t e s t c o u l d n o t b e u s e d w i t h c r u d e s a p b e c a u s e o f n o n -56 T a b l e 4 . C o m p a r i s o n o f i n f e c t e d / h e a l t h y a b s o r b a n c e r a t i o i n d o u b l e a n t i b o d y s a n d w i c h E L I S A f o r c r o s s - a d s o r b e d v e r s u s n o n c r o s s - a d s o r b e d a p p l e c h l o r o t i c l e a f s p o t v i r u s a n t i s e r u m . A n t i s e r u m V c o n j u g a t e d i l u e n t b I n f e c t e d / H e a l t h y R a t i o 0 A U n a d s o r b e d / P T B 1 . 2 9 A U n a d s o r b e d / s a p 1 4 . 5 0 A A d s o r b e d / P T B 1 . 5 5 A A d s o r b e d / s a p 5 . 0 8 B U n a d s o r b e d / P T B 1 . 7 0 B U n a d s o r b e d / s a p 7 .52 B A d s o r b e d / P T B 2 .25 B A d s o r b e d / s a p 3 . 2 0 a A a n d B r e f e r t o t h e two i s o l a t e s from P r u n u s g l a n d u l o s a Conjugate d i l u t e d either i n P B S , 0.05% Tween, 0.1% s k i m m i l k p o w d e r (PTB) o r h o s t s a p . I n f e c t e d a n d h e a l t h y C h e n o p o d i u m q u i n o a g r o u n d a t 1 : 1 0 t i s s u e t o b u f f e r r a t i o . c R a t i o s w e r e d e t e r m i n e d f r o m t h e a v e r a g e o f e i g h t w e l l s / t r e a t m e n t 57 • ® F i g . 1 0 . A g a r g e l d o u b l e d i f f u s i o n t e s t w i t h p u r i f i e d a p p l e c h l o r o t i c l e a f s p o t v i r u s i s o l a t e s A a n d B . A s = i s o l a t e B p o l y c l o n a l a n t i s e r u m . W e l l s B a n d E = i s o l a t e A . W e l l s C a n d F = i s o l a t e B . W e l l A = H e a l t h y C h e n o p o d i u m q u i n o a . W e l l D = b u f f e r o n l y . 58 s p e c i f i c host r e a c t i o n s . The technique of d i l u t i n g conjugate i n sap worked w e l l , however, f r e s h C. quinoa leaves may not be a v a i l a b l e so a comparison was made using frozen sap and sap st o r e d at 4 C with 0.02% sodium a z i d e . Results w i t h f r e s h or sto r e d sap were equ i v a l e n t but i n f e c t e d / h e a l t h y r a t i o s were reduced from 12 and 13.5 r e s p e c t i v e l y t o 2.5 with frozen sap. The r a t i o was a l s o lower when the PTB b u f f e r was supplemented w i t h MgS04 or TM b u f f e r was used (Table 5). I n i t i a l f u s i o n s done a f t e r i n j e c t i n g mice wi t h whole v i r u s f a i l e d t o produce any v i r u s - s p e c i f i c hybridomas. When the v i r u s was d i s r u p t e d with 1% SDS before i n j e c t i o n , s i x p o s i t i v e clones out of 480 screened were detected. Further t e s t i n g r e vealed that two of the s i x clones could detect the v i r u s when coated d i r e c t l y t o the polyst y r e n e p l a t e but not i n i n f e c t e d C. quinoa sap while the four other clones could detect the v i r u s under both c o n d i t i o n s . A f t e r r e c l o n i n g , these four hybridomas were r e t e s t e d against seven i s o l a t e s of ACLSV (Table 6). Clones A2, C3 and D4 reacted w i t h the Prunus i s o l a t e s only whereas clone B2 detected a l l i s o l a t e s but d i d not give as strong a r e a c t i o n as the other three clones. The subclass of immunoglobulins secreted by the hybridomas was IgGl f o r clones A2, C3 and D4 and IgG2a f o r clone B2. In a TAS-ELISA the B2 MAB was d i l u t e d i n t w o - f o l d steps from 1/100 t o 1/51200. When l e f t overnight, the absorbance values f o r T a b l e 5 . C o m p a r i s o n o f t h r e e g r i n d i n g b u f f e r s a n d t h r e e m e t h o d s o f d i l u t i n g c o n j u g a t e on t h e a b s o r b a n c e r a t i o o f i n f e c t e d / h e a l t h y C . q u i n o a i n d o u b l e a n t i b o d y s a n d w i c h E L I S A . Sap T y p e a B u f f e r 1 3 F r e s h F r o z e n S t o r e d A 1 2 . 0 C 2 . 5 1 3 . 5 B 8 . 5 4 . 7 1 0 . 9 C 1 0 . 0 3 . 4 8 . 8 a S a p T y p e - ( g r o u n d 1 : 1 0 i n PTB) F r e s h = H e a l t h y C . q u i n o a s a p F r o z e n = H e a l t h y C . q u i n o a s a p s t o r e d a t - 2 0 C S t o r e d = H e a l t h y C . q u i n o a s a p s t o r e d a t 4 C b B u f f e r A = P B S , 0.05% T w e e n - 2 0 , 0.1% B l o t t o ( s k i m m i l k p o w d e r ) (PTB) B = P T B , 0 . 0 1 M M g S 0 4 C = 0 . 0 5 M T r i s , 0 . 0 1 M M g S 0 4 , 0.1% B l o t t o , 0.05% T w e e n - 2 0 c R a t i o s w e r e d e t e r m i n e d f r o m t h e a v e r a g e o f t h r e e w e l l s / t r e a t m e n t 60 Table 6. Reactions of fo u r monoclonal a n t i b o d i e s (MAB) to seven i s o l a t e s of apple c h l o r o t i c l e a f s p o t v i r u s i n Chenopodium quinoa by t r i p l e antibody sandwich ELISA Mean A.n. observed with MAB I s o l a t e A2 B2 C3 D4 B (Prunus) 0 . 640a 0 . 190 0 . 490 0. .450 A (Prunus) 0 , . 670 0 .250 0 . 470 0 .710 ATCC (Malus) 0 . 028 0 , .210 0 .017 0 .071 P205 (Malus) 0 . 031 0 .260 0 .019 0 . 058 UBC (Malus) 0 , .034 0 .210 0 .020 0 . 047 SI (Prunus) 0 , .420 0 .300 0 .280 0 .420 1000-04 (Malus) 0 , .030 0 , .230 0 .017 0 .061 Healthy C. quinoa 0 , .010 0 . 006 0 .005 0 .009 aAverage of t h r e e w e l l s i n f e c t e d C . q u i n o a a t t h e 1 /51200 d i l u t i o n w e r e b e t w e e n 0 . 5 0 a n d 0 . 9 4 a n d t h e h e a l t h y v a l u e s w e r e b e t w e e n 0 . 0 0 3 a n d 0 . 0 2 4 . A b u f f e r c o n t a i n i n g P T B p l u s 2% P V P , 2.5% n i c o t i n e a n d 0 . 0 1 M M g S 0 4 was t h e b e s t f o r d e t e c t i n g t h e h o m o l o g o u s a n t i g e n f r o m woody a n d h e r b a c e o u s h o s t s ( T a b l e 7 ) . A l t h o u g h t h e B2 MAB c o u l d d e t e c t a l l s e v e n i s o l a t e s i n T A S -E L I S A i t c o u l d n o t b e u s e d as a t r a p p i n g a n t i b o d y i n D A S - E L I S A . H o w e v e r , B2 c o u l d b e c o n j u g a t e d d i r e c t l y t o a l k a l i n e p h o s p h a t a s e a n d u s e d w i t h a p o l y c l o n a l t r a p p i n g a n t i b o d y . I n s u c h t e s t s t h e a b s o r b a n c e v a l u e s w e r e h i g h e r t h a n when t h e same MAB was u s e d i n T A S - E L I S A w i t h no i n c r e a s e i n h e a l t h y b a c k g r o u n d r e a c t i o n s . 62 T a b l e 7 . T h e e f f e c t s o f s i x d i f f e r e n t g r i n d i n g b u f f e r a d d i t i v e s on t h e d e t e c t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s i s o l a t e A i n C h e n o p o d i u m q u i n o a a n d P r u n u s g l a n d u l o s a b y t r i p l e a n t i b o d y s a n d w i c h E L I S A A 4 0 5 o b s e r v e d w i t h a d d i t i v e s T i s s u e S o u r c e 1 2 3 4 5 6 C . q u i n o a 0 , . 7 5 7 b 0 .  990 0 . 375 0 . . 123 1 , .221 1. .885 P . g l a n d u l o s a 0 . . 2 2 8 b 0 . .119 0. . 109 0. .097 0 . .181 0 . .350 H e a l t h y C . q u i n o a 0 . . 0 0 1 c 0 . .015 0 . .010 0 . .006 0 , .002 0 . .003 B u f f e r o n l y 0 . 0 1 2 c 0 . 003 0 . 001 0 . .008 0 , .003 0 . . 002 a T h e s t a n d a r d b u f f e r was PBS w i t h 0.05% T w e e n - 2 0 , 0.1% s k i m m i l k p o w d e r , 2% P V P . V a r i o u s c o m p o u n d s w e r e a d d e d as f o l l o w s : 1 = no a d d i t i v e s ; 2 = N i c o t i n e (2 .5%) ; 3 = M g S 0 4 ( 0 . 0 1 M ) ; 4 = d i a m i n o -d i p r o p y l a m i n e (DD) ( 0 . 0 2 % ) ; 5 = N i c o t i n e , M g S 0 4 , D D ; 6 = N i c o t i n e , M g S 0 4 b A v e r a g e o f t h r e e w e l l s ° S i n g l e w e l l o n l y 63 D i s c u s s i o n T h e A C L S V p o l y c l o n a l a n t i s e r u m h a d a h i g h p r o p o r t i o n o f a n t i b o d i e s d i r e c t e d a g a i n s t h o s t c o m p o n e n t s a n d w i t h o u t c r o s s -a d s o r p t i o n c o u l d n o t d i s c r i m i n a t e b e t w e e n i n f e c t e d a n d h e a l t h y t i s s u e . T h e i n f e c t e d / h e a l t h y r a t i o s l i s t e d i n T a b l e 4 show t h a t d i l u t i n g t h e a n t i b o d y - e n z y m e c o n j u g a t e i n h o s t s a p i s t h e m o s t e f f e c t i v e way o f r e m o v i n g n o n - s p e c i f i c r e a c t i o n s . F r e s h l y p r e p a r e d h o s t s a p o r s a p s t o r e d a t 4 C c o u l d b e u s e d t o d i l u t e t h e c o n j u g a t e b u t n o t p r e v i o u s l y f r o z e n s a p . F r e e z i n g h a s b e e n u s e d a s a c l a r i f i c a t i o n t e c h n i q u e f o r p r e c i p i t a t i o n o f p l a n t p r o t e i n s d u r i n g v i r u s p u r i f i c a t i o n . T h e r e f o r e , t h e r e d u c e d i n f e c t e d / h e a l t h y r a t i o s e e n when p r e v i o u s l y f r o z e n s a p was u s e d a s t h e d i l u e n t ( T a b l e 5) p r o b a b l y r e s u l t e d f r o m t h e p r e c i p i t a t i o n o f h o s t p r o t e i n s n e e d e d f o r c r o s s - a d s o r p t i o n . T h e more c o m m o n l y u s e d t e c h n i q u e o f c r o s s - a d s o r b i n g t h e raw a n t i s e r u m b e f o r e p u r i f i c a t i o n o f g a m m a - g l o b u l i n s r e s u l t e d i n a l o w e r r a t i o , p r o b a b l y d u e t o n o n - s p e c i f i c r e m o v a l o f a n t i b o d i e s a g a i n s t t h e v i r u s . O t h e r a u t h o r s (2 ,18 ) h a v e r e p o r t e d h i g h b a c k g r o u n d r e a c t i o n s w i t h A C L S V a n t i s e r u m a n d t h e n e e d f o r c r o s s - a d s o r p t i o n . T h e A a n d B i s o l a t e s a r e s e r o l o g i c a l l y i d e n t i c a l a l t h o u g h t h e y d i f f e r s l i g h t l y i n symptoms c a u s e d on h e r b a c e o u s h o s t s ( see C h a p t e r 2 ) . I n a g a r g e l d i f f u s i o n t e s t s , a c o n t i n u o u s p r e c i p i t i n l i n e was f o r m e d b e t w e e n t h e two i s o l a t e s ( F i g . 10) a n d t h e y r e a c t e d i d e n t i c a l l y i n t h e E L I S A t e s t . T h e i n i t i a l f u s i o n s f o r MAB p r o d u c t i o n w e r e d o n e u s i n g w h o l e v i r u s a n d r e s u l t e d i n c l o n e s s p e c i f i c f o r h o s t c o m p o n e n t s o n l y . B e c a u s e o f t h e v i r u s m o r p h o l o g y , t h e p a r t i c l e s f r e q u e n t l y become e n t a n g l e d w i t h t h e m s e l v e s a n d h o s t m a t e r i a l t o f o r m l a r g e a g g r e g a t e s w h i c h a r e d i f f i c u l t t o s e p a r a t e . When i n j e c t e d i n t o t h e m o u s e , s u c h a g g r e g a t e s w o u l d i l l i c i t a h i g h r e s p o n s e t o h o s t a n t i g e n s . When v i r u s d i s r u p t e d w i t h SDS was u s e d f o r i m m u n i z a t i o n , s i x p o s i t i v e c l o n e s o u t o f 480 s c r e e n e d w e r e d e t e c t e d a n d f e w e r c l o n e s a g a i n s t h o s t a n t i g e n s w e r e p r o d u c e d . W h e t h e r t h i s s u c c e s s was due t o i n j e c t i o n o f d i s r u p t e d v i r u s i s n o t k n o w n . I f o n l y a s m a l l p e r c e n t a g e o f v i r u s - s p e c i f i c h y b r i d o m a s a r e p r o d u c e d (6 /480 = 1.25%) t h e s e may h a v e b e e n m i s s e d d u r i n g t h e s c r e e n i n g p r o c e s s a f t e r i n j e c t i o n o f w h o l e v i r u s as n o t a l l h y b r i d o m a s a r e s t a b l e a n d s u r v i v e . M o s t MABs a r e c o n f o r m a t i o n - s p e c i f i c a n d r e c o g n i z e s e p a r a t e r e g i o n s o f a m i n o a c i d s e q u e n c e b r o u g h t t o g e t h e r b y t h e f o l d i n g o f t h e p o l y p e p t i d e c h a i n s ( 7 7 ) . The f o u r MABs c a p a b l e o f d e t e c t i n g t h e v i r u s i n s a p w e r e s e p a r a t e d i n t o two c a t e g o r i e s , t h o s e t h a t d e t e c t o n l y s t r a i n s f r o m P r u n u s (A2, C 3 , D4) a n d t h o s e t h a t d e t e c t a l l s t r a i n s u s e d i n t h i s s t u d y ( B 2 ) . T h e c l o n e s s p e c i f i c f o r t h e P r u n u s s t r a i n s g a v e a much s t r o n g e r r e a c t i o n t h a n t h e b r o a d s p e c t r u m c l o n e a n d p r o b a b l y a r e d i r e c t e d a g a i n s t a c o n f o r m a t i o n a l e p i t o p e o n t h e s u r f a c e o f t h e v i r i o n w h i l e t h e b r o a d s p e c t r u m c l o n e may b e s p e c i f i c f o r an i n t e r n a l e p i t o p e common t o a l l s t r a i n s . T h i s i s s u p p o r t e d b y t h e f a c t t h a t t h e B2 c l o n e c a n n o t b e u s e d as a t r a p p i n g a n t i b o d y i n E L I S A . To v e r i f y t h i s , t h e e f f e c t s o f a d d i n g s u b s t a n c e s s u c h a s SDS t o t h e g r i n d i n g b u f f e r w h i c h w o u l d d e n a t u r e t h e v i r u s a n d e x p o s e 65 i n t e r n a l e p i t o p e s s h o u l d b e i n v e s t i g a t e d . O f t h e f i v e g r i n d i n g b u f f e r a d d i t i v e s i n v e s t i g a t e d t h e b e s t r e s u l t s w e r e o b t a i n e d w i t h t h e s t a n d a r d b u f f e r (PTB w i t h 2% PVP) s u p p l e m e n t e d w i t h 2.5% n i c o t i n e a n d 0 . 0 1 M M g S 0 4 ( T a b l e 7 ) . T h i s r e s e a r c h i n d i c a t e s t h a t a m o n o c l o n a l a n t i b o d y ( c l o n e B2) may b e u s e f u l a s a b r o a d s p e c t r u m d e t e c t i o n t o o l f o r t h e many d i f f e r e n t b i o l o g i c a l s t r a i n s o f A C L S V i n f r u i t t r e e s , h o w e v e r , f u r t h e r c h a r a c t e r i z a t i o n o f t h e MABs m u s t s t i l l b e d o n e . F o r i n s t a n c e , t h e B2 c l o n e s h o u l d b e c h e c k e d f o r h e t e r o s p e c i f i c r e a c t i o n s a g a i n s t o t h e r c l o s t e r o v i r u s e s . A l l c l o n e s s h o u l d b e t e s t e d a g a i n s t more A C L S V i s o l a t e s i n woody h o s t s , e s p e c i a l l y t h o s e t h a t a r e d i f f i c u l t t o d e t e c t v i r u s f r o m , s u c h a s p e a r a n d a p r i c o t . D i f f e r e n t t y p e s o f woody h o s t t i s s u e s s u c h as l e a v e s , b l o s s o m s , c a m b i u m , a n d r o o t s c o u l d b e i n v e s t i g a t e d . A l s o , t h e b r o a d s p e c t r u m a n d P r u n u s - s p e c i f i c c l o n e s c o u l d b e c o m p a r e d i n c o m p e t i t i v e i n h i b i t i o n a s s a y s a n d p o s s i b l y u s e d t o s t u d y a n t i g e n i c v a r i a t i o n i n A C L S V s t r a i n s . C h a p t e r 6 - I s o l a t i o n o f T o b a c c o M o s a i c V i r u s  I n t r o d u c t i o n T o b a c c o m o s a i c v i r u s (TMV) i s o c c a s i o n a l l y f o u n d a s a l a t e n t i n f e c t i o n i n f r u i t t r e e s , h o w e v e r , no symptoms h a v e e v e r b e e n o b s e r v e d o n woody i n d i c a t o r s ( 6 0 ) . I n f e c t e d t r e e s show no symptoms a n d i n s w e e t c h e r r y t h e v i r u s c o n c e n t r a t i o n i s s o l o w t h a t a t t e m p t s t o i s o l a t e TMV f r o m known i n f e c t e d p l a n t s o f t e n f a i l e d ( 3 0 ) . I n H u n g a r y , B u r g y a n e t a l . (7) i s o l a t e d TMV f r o m p l u m w h i c h was s e r o l o g i c a l l y i d e n t i c a l t o T M V - U 1 b u t d i f f e r e d s l i g h t l y i n s y m p t o m o l o g y o n h e r b a c e o u s h o s t s . T h e p l u m i s o l a t e o c c a s i o n a l l y p r o d u c e d a s y s t e m i c l a t e n t i n f e c t i o n i n C . q u i n o a a n d p r o d u c e d a s t e m n e c r o s i s i n N i c o t i a n a g l u t i n o s a L . M a t e r i a l s a n d M e t h o d s T h e b u f f e r u s e d f o r t r a n s f e r o f t h e TMV i s o l a t e s was 0 . 0 5 M s o d i u m p h o s p h a t e , p H 7 . 5 , c o n t a i n i n g 2% p o l y v i n y l p y r o l i d o n e ( P V P ) . F o r i s o l a t i o n s f r o m woody h o s t l e a v e s , f l o w e r s a n d r o o t s , 1.0% n i c o t i n e was a d d e d t o t h e b u f f e r s a n d t h e p l a n t s w e r e r i n s e d w i t h w a t e r a f t e r b e i n g i n o c u l a t e d . T h e f o l l o w i n g h e r b a c e o u s p l a n t s w e r e t e s t e d w i t h t h e TMV i s o l a t e s : C h e n o p o d i u m a m a r a n t i c o l o r C o s t e & R e y n . , C . q u i n o a , C u c u m i s s a t i v a L . ^ S t r a i g h t 8 ' , D a t u r a s t r a m o n i u m L . , N i c o t i a n a b e n t h a m i a n a D o m i n . , N i c o t i a n a c l e v l a n d i i G r a y , N . g l u t i n o s a , N i c o t i a n a t a b a c u m L . ^ H a r r o w n o v a ' , x X a n t h i ' , ^ S a m s u n ' , N i c o t i a n a r u s t i c a L . , V i g n a u n g u i c u l a t a ( L . ) W a l p . , a n d Z i n n i a e l e g a n s J a c q . T M V - 1 2 9 2 was p u r i f i e d f r o m N . b e n t h a m i a n a . T h e t i s s u e was 67 h o m o g e n i z e d i n two v o l u m e s o f 0 . 0 5 M s o d i u m p h o s p h a t e pH 7 . 5 a n d c l a r i f i e d w i t h 8.0% b u t a n o l . T h e s u p e r n a t a n t was p r e c i p i t a t e d w i t h 8.0% p o l y e t h y l e n e g l y c o l (PEG) a n d 1.0% N a C l , r e s u s p e n d e d i n 0 . 1 v o l u m e p h o s p h a t e b u f f e r a n d g i v e n one l o w s p e e d c e n t r i f u g a t i o n o f 20 m i n a t 1 4 , 0 0 0 g . T h e s a m p l e was c e n t r i f u g e d a t 1 1 1 , 0 0 0 g f o r 90 m i n a n d r e s u s p e n d e d i n 4 . 0 m l o f b u f f e r f o l l o w e d b y i s o p y c n i c c e n t r i f u g a t i o n i n 40% c a e s i u m c h l o r i d e f o r 16 h r a t 1 7 1 , 0 0 0 g . T h e v i r a l b a n d was w i t h d r a w n a n d d i a l y s e d a g a i n s t p h o s p h a t e b u f f e r . P r o d u c t i o n o f a n t i s e r u m t o T M V - 1 2 9 2 was as d e s c r i b e d i n C h a p t e r 5 f o r A C L S V . R e s u l t s T h e o r i g i n a l i s o l a t i o n o f T M V - 1 2 9 2 was f r o m t h e s y s t e m i c l e a v e s o f i s o l a t e B A C L S V i n f e c t e d C . q u i n o a t o one o f f o u r N . b e n t h a m n i a n a . F u r t h e r t e s t s f r o m P . g l a n d u l o s a w e r e d o n e i n w h i c h s i x o f s i x N . b e n t h a m n i a n a became i n f e c t e d b u t no l o c a l l e s i o n s a p p e a r e d on N . t a b a c u m ' X a n t h i ' . A f t e r one more s u c c e s s f u l i s o l a t i o n s u b s e q u e n t a t t e m p t s f a i l e d t o d e t e c t a n y T M V - 1 2 9 2 . To t e s t i f t h e s y s t e m i c ' h o s t was a c t u a l l y more s e n s i t i v e t h a n t h e l o c a l l e s i o n h o s t , a s e r i e s o f 1 0 - f o l d d i l u t i o n s o f p u r i f i e d T M V -1292 f r o m 1 m g / m l t o 1 n g / m l w e r e r u b i n o c u l a t e d t o t h e l o c a l l e s i o n h o s t N . g l u t i n o s a . The d i l u t i o n e n d p o i n t was 10 n g / m l . S u b s e q u e n t l y , f o u r N . g l u t i n o s a a n d f o u r N . b e n t h a m n i a n a w e r e i n o c u l a t e d w i t h 10 n g / m l a n d 1 n g / m l p u r i f i e d v i r u s . A t t h e h i g h e r c o n c e n t r a t i o n one o f f o u r N . g l u t i n o s a d e v e l o p e d l e s i o n s 68 w h i l e t h r e e o f f o u r N . b e n t h a m n i a n a became i n f e c t e d . A t t h e l o w e r c o n c e n t r a t i o n , no N . g l u t i n o s a w e r e i n f e c t e d a n d one o f f o u r N . b e n t h a m n i a n a d e v e l o p e d s y m p t o m s . The i n f e c t e d p l a n t s f r o m b o t h d i l u t i o n s w e r e b a c k i n o c u l a t e d t o N . g l u t i n o s a a n d N . b e n t h a m i a n a a n d a l l p l a n t s d e v e l o p e d t y p i c a l TMV s y m p t o m s . T h e h e r b a c e o u s h o s t r a n g e o f T M V - 1 2 9 2 i s c o n s i s t e n t w i t h o t h e r TMV i s o l a t e s ( T a b l e 8 ) . T h e c o a t p r o t e i n o f t h e T M V - 1 2 9 2 was e s t i m a t e d a t 1 7 . 9 KDa a n d m i g r a t e d i d e n t i c a l l y t o T M V - U 1 on t h e same g e l . When d s R N A was e x t r a c t e d f r o m T M V - 1 2 9 2 i n f e c t e d N . b e n t h a m n i a n a t h e MW o f t h e m a j o r b a n d was 4 . 3 x 10 6 d a l t o n s w i t h m i n o r b a n d s a t 2 . 4 x 1 0 6 a n d 0 . 8 x 1 0 6 d a l t o n s . T h e s s R N A was e s t i m a t e d a t 2 . 0 7 x 1 0 6 d a l t o n s a n d m i g r a t e d a t t h e same r a t e a s T M V - U 1 RNA o n t h e same g e l . T h e T M V - 1 2 9 2 a n t i s e r u m d e t e c t e d t h e h o m o l o g o u s a n d U l i s o l a t e s i n N i c o t i a n a s p e c i e s b y E L I S A a n d p u r i f i e d v i r u s b y E L I S A a n d a g a r g e l d i f f u s i o n . R e p e a t e d a t t e m p t s t o d e t e c t TMV i n P . g l a n d u l o s a f a i l e d a s d i d a t t e m p t s t o r e - i s o l a t e t h e v i r u s f r o m t h e woody h o s t a f t e r t h e f i r s t two s u c c e s s f u l i s o l a t i o n s . T a b l e 8 . T h e h e r b a c e o u s h o s t r a n g e o f t o b a c c o m o s a i c v i r u s (TMV) i s o l a t e d f r o m P r u n u s g l a n d u l o s a . S p e c i e s Symptoms' 1 C h e n o p o d i u m a m a r a n t i c o l o r C . q u i n o a C u c u m i s s a t i v a D a t u r a s t r a m o n i u m N i c o t i a n a b e n t h a m i a n a N . c l e v l a n d i i N . g l u t i n o s a N . r u s t i c a N . t a b a c u m ^ H a r r o w n o v a ' N . t a b a c u m H a m s u n ' N . t a b a c u m ^ X a n t h i ' V i g n a u n g u i c u l a t a Z i n n i a e l e g a n s n e c 11 n e c 11 1 1 , s y s d i s t 11 s t u n t s y s l e s 11 s y s l e s s y s l e s s y s l e s 11 0 11 a K e y : 11 = l o c a l l e s i o n s ; s y s = s y s t e m i c ; l e s = l e s i o n s ; d i s t d i s t o r t i o n ; n e c = n e c r o t i c ; 0 = no i n f e c t i o n 7 0 D i s c u s s i o n T h e h o s t r a n g e f o r T M V - 1 2 9 2 d i f f e r e d f r o m t h e t y p e s t r a i n r a n g e ( 3 8 ) o n l y i n t h e r e a c t i o n on C . s a t i v a ( l o c a l l e s i o n s a n d s y s t e m i c d i s t o r t i o n ) . T h e u n i q u e symptoms i n N . g l u t i n o s a r e p o r t e d b y B u r g y a n e t a l . ( 7 ) f o r a p l u m i s o l a t e o f TMV w e r e n o t o b s e r v e d . TMV i s known t o g i v e r i s e t o a w i d e r a n g e o f m u t a n t t y p e s s o t h i s i s n o t u n u s u a l ( 8 3 ) . When i s o l a t i n g v i r u s e s f r o m woody h o s t s , t h e v i r u s may b e m i s s e d b e c a u s e o f l o w t i t r e o r may o n l y p r o d u c e a f e w l e s i o n s . I n s u c h c a s e s i t i s n e c e s s a r y t o b u i l d u p t h e t i t r e b y a p a s s a g e t h r o u g h t h e h e r b a c e o u s h o s t b e f o r e a t t e m p t i n g t o d e t e r m i n e t h e h o s t r a n g e . T h i s f a c t was d e m o n s t r a t e d w i t h t h e i s o l a t i o n o f TMV f r o m P . g l a n d u l o s a when t h e u s u a l l o c a l l e s i o n i n d i c a t o r s w e r e n o t i n f e c t e d . The p o s s i b i l i t y o f TMV c o n t a m i n a t i o n c a n b e r u l e d o u t b e c a u s e e x t r e m e p r e c a u t i o n s w e r e t a k e n a n d n o n e o f t h e c o n t r o l p l a n t s s h o w e d a n y e v i d e n c e o f i n f e c t i o n . The b e s t e x p l a n a t i o n i s t h a t t h e TMV o c c u r s i n a l o w c o n c e n t r a t i o n i n P . g l a n d u l o s a a n d / o r i s i r r e g u l a r l y d i s t r i b u t e d as i n c h e r r y ( 3 0 ) . T h e p h y s i c a l a n d s e r o l o g i c a l c h a r a c t e r i s t i c s o f T M V - 1 2 9 2 w e r e c l o s e t o t h o s e p r e v i o u s l y r e p o r t e d ( 8 3 , 1 6 ) f o r a t o b a c c o i s o l a t e o f T M V . 71 C h a p t e r 7 - G e n e r a l D i s c u s s i o n I n g e n e r a l t h e b i o l o g i c a l , p h y s i c a l a n d s e r o l o g i c a l p r o p e r t i e s o f t h e two A C L S V i s o l a t e s s t u d i e d w e r e t h e same a s t h o s e p r e v i o u s l y r e p o r t e d a n d c o n f i r m t h e d i a g n o s i s o f t h e i s o l a t e s as A C L S V . Some d i f f e r e n c e s w e r e n o t e d i n s y m p t o m o l o g y on h e r b a c e o u s a n d woody i n d i c a t o r s . I s o l a t e A d i f f e r e d f r o m t h e o t h e r A C L S V i s o l a t e s b y n o t m o v i n g s y s t e m i c a l l y i n C . a m a r a n t i c o l o r a n d b o t h A a n d B i s o l a t e s f a i l e d t o i n f e c t some s p e c i e s r e p o r t e d a s s u s c e p t i b l e b y o t h e r a u t h o r s . F u r t h e r , t h e l a c k o f symptoms on t h e woody i n d i c a t o r s was u n e x p e c t e d . T h i s v a r i a b i l i t y h a s b e e n r e p o r t e d p r e v i o u s l y (56 ,21 ) a n d c a n b e e x p e c t e d f o r a v i r u s t h a t c a u s e s s u c h a w i d e s p e c t r u m o f d i s e a s e s i n i t ' s n u m e r o u s h o s t s (17) . C l o s t e r o v i r u s e s a r e among t h e m o s t d i f f i c u l t v i r u s e s t o p u r i f y . Y i e l d s a r e t y p i c a l l y l o w , a s t h e v i r u s e s a r e p h l o e m l i m i t e d , a n d t h e p a r t i c l e s f r e q u e n t l y d a m a g e d . T h e p a r t i c l e s a l s o a g g r e g a t e a n d a r e a d s o r b e d t o h o s t membranes ( 3 ) . T h e p u r i f i c a t i o n m e t h o d d e s c r i b e d i n t h i s p a p e r i s s i m p l e a n d n o t as d e p e n d e n t on t h e i n i t i a l c l a r i f i c a t i o n s t e p as a r e o t h e r A C L S V p u r i f i c a t i o n m e t h o d s . The E L I S A v a l u e s f o r t h e p u r i f i c a t i o n p e l l e t s a n d s u p e r n a t a n t show t h a t a c o n s i d e r a b l e amount o f v i r u s i s l o s t d u r i n g l o w s p e e d c e n t r i f u g a t i o n . I f s u i t a b l e c o n d i t i o n s o f i o n i c s t r e n g t h o r m o l a r i t y c o u l d b e f o u n d u n d e r w h i c h t h e p a r t i c l e a d s o r p t i o n t o h o s t t i s s u e was d e c r e a s e d , t h e v i r u s y i e l d c o u l d b e s u s t a n t i a l l y i n c r e a s e d . The s e c o n d a r e a o f A C L S V p u r i f i c a t i o n n e e d i n g i m p r o v e m e n t i s t h e e l i m i n a t i o n o f t h e 72 c o n t a m i n a t i n g s p h e r i c a l s t r u c t u r e s r e s p o n s i b l e f o r t h e s e r o l o g i c a l r e a c t i o n s t o h o s t t i s s u e o b s e r v e d w i t h t h e p o l y c l o n a l a n t i s e r a . T h e s p h e r e s c o u l d n o t b e p u r i f i e d b y t h e A C L S V m e t h o d f r o m h e a l t h y C . q u i n o a a n d may b e p r e s e n t i n l a r g e n u m b e r s o n l y i n v i r u s - i n f e c t e d o r s t r e s s e d p l a n t s . I f an a n t i s e r u m s p e c i f i c f o r t h e s p h e r e s w e r e p r o d u c e d , t h e y may b e e l i m i n a t e d b y c r o s s -a d s o r p t i o n . T h i s s t u d y a g r e e s w i t h p r e v i o u s r e p o r t s t h a t t h e t y p i c a l b e e t y e l l o w s t y p e o f v e s i c l e s f o u n d a s s o c i a t e d w i t h s u b g r o u p I c l o s t e r o v i r u s e s a r e n o t p r e s e n t i n A C L S V i n f e c t e d t i s s u e ( 2 5 ) . T h e p a r t i c l e l e n g t h , w i d t h a n d p i t c h a r e a l s o s i m i l a r t o t h o s e p r e v i o u s l y r e p o r t e d (4 ,72 ) . U n f o r t u n a t e l y , t h e l o n g f l e x u o u s r o d s s e e n i n t h e v a s c u l a r e l e m e n t s c o u l d n o t b e d i s t i n g u i s h e d f r o m P -p r o t e i n . A s p a r t o f t h e f u r t h e r c h a r a c t e r i z a t i o n o f t h e m o n o c l o n a l a n t i b o d i e s , t h e i r a b i l i t i y t o b i n d t o i n t a c t v i r i o n s a n d r e a c t w i t h p r o t e i n - A c o l l o i d a l g o l d m a r k e r s h o u l d b e i n v e s t i g a t e d . T h i s t e c h n i q u e w o u l d a l l o w f o r a d e f i n i t i v e i d e n t i f i c a t i o n o f t h e f l e x u o u s r o d s i n t h e s e i v e t u b e e l e m e n t s a n d f a c i l i t a t e f u r t h e r s t u d i e s on A C L S V c y t o p a t h o l o g y , e s p e c i a l l y i n l i g h t o f t h e r e c e n t r e p o r t o f A C L S V p a r t i c l e s i n t h e m e s o p h y l l o f C . q u i n o a (61) . A s m o r e r e s e a r c h i s d o n e on t h e c l o s t e r o v i r u s e s , i t i s c l e a r t h a t t h e g r o u p i s n o t h o m o g e n e o u s , b u t made u p o f a n u m b e r o f s u b g r o u p s w h i c h a r e r e l a t e d o n l y on t h e b a s i s o f g r o s s m o r p h o l o g y . A C L S V d i f f e r s f r o m t h e t y p e member b e e t y e l l o w s i n p a r t i c l e l e n g t h , c y t o p a t h o l o g y ( l a c k o f v e s i c l e s , n o t p h l o e m 73 l i m i t e d ) , no a p h i d v e c t o r , s a p t r a n s m i s s i o n a n d d s R N A p a t t e r n ( 2 5 , 1 9 , 8 1 , 6 1 ) a n d w i l l p r o b a b l y b e s e p a r a t e d f r o m t h e c l o s t e r o v i r u s e s i n t h e f u t u r e as more i n f o r m a t i o n i s g a t h e r e d on t h e genome o r g a n i z a t i o n o f g r o u p m e m b e r s . T h e s e r o l o g i c a l r e s u l t s w i t h t h e p o l y c l o n a l a n t i s e r a e m p h a s i z e t h e p r o b l e m s e n c o u n t e r e d w i t h a n t i s e r a p r o d u c e d f r o m c o n t a m i n a t e d v i r u s p r e p a r a t i o n s . I n t h i s c a s e t h e c o n t a m i n a t i n g p r o t e i n was d i f f i c u l t t o e l i m i n a t e a n d t h e a n t i s e r u m n e e d e d c r o s s - a d s o r p t i o n b y d i l u t i n g t h e e n z y m e - c o n j u g a t e i n h o s t s a p . R e s u l t s f o r A C L S V d e t e c t i o n i n P . g l a n d u l o s a b y t h i s m e t h o d w e r e v a r i a b l e , p r o b a b l y d u e t o i r r e g u l a r v i r u s d i s t r i b u t i o n (26) o r l o w v i r u s t i t r e . A c e r t i f i c a t i o n p r o g r a m b a s e d s o l e l y on s e r o l o g i c a l t e c h n i q u e s u s i n g p o l y c l o n a l a n t i s e r u m w o u l d c l e a r l y b e i n a d e q u a t e u n l e s s e x t e n s i v e t e s t i n g o f t h e m o s t s u i t a b l e t y p e o f h o s t t i s s u e , t h e o p t i m u m t i m e o f y e a r f o r s a m p l i n g a n d t h e r e a c t i o n s a g a i n s t t h e v a r i o u s s t r a i n s w e r e d o n e . G r u n t z i g a n d F u c h s (34) f o u n d t h a t u n l e s s t h e E L I S A t e s t was d o n e u s i n g p e t a l s i n A p r i l t o M a y , a b i o l o g i c a l t e s t on C . q u i n o a s h o u l d b e u s e d t o e n s u r e a 90% r e l i a b i l i t y r a t e . M o n o c l o n a l a n t i b o d i e s o f f e r i n c r e a s e d d e t e c t i o n s e n s i t i v i t y o v e r p o l y c l o n a l a n t i b o d i e s a n d g i v e l o w b a c k g r o u d l e v e l s . W i t h t h e a p p r o p r i a t e s c r e e n i n g m e t h o d s , c l o n e s c a n b e s e l e c t e d w i t h v a r y i n g d e g r e e s o f s e r o l o g i c a l s p e c i f i c i t y ( 5 7 ) . P o u l a n d D u n e z (66) p r o d u c e d two m o n o c l o n a l l i n e s w h i c h r e c o g n i z e d 17 A C L S V i s o l a t e s f r o m P r u n u s a n d M a l u s . T h e i r E L I S A t e s t u s e d one MAB f o r c o a t i n g a n d t h e s e c o n d was l a b e l l e d w i t h a l k a l i n e p h o s p h a t a s e . 74 These t y p e s o f e x p e r i m e n t s must s t i l l be done w i t h t h e i s o l a t e A m o n o c l o n a l s . The u l t i m a t e g o a l i s t o d e v i s e an ELISA t e c h n i q u e f o r r o u t i n e d e t e c t i o n o f ACLSV from woody p l a n t s and e v a l u a t e t h e optimum t i s s u e t y p e and t i m e o f y e a r f o r s a m p l i n g . The B2 MAB i s t h e o n l y one w i t h p o t e n t i a l f o r t h i s t y p e o f a p p l i c a t i o n u n l e s s t h e t e s t s were l i m i t e d t o Prunus s p e c i e s . Other a r e a s o f r e s e a r c h such as v i r u s e l i m i n a t i o n , v e c t o r r e l a t i o n s h i p s and s u r v e y s c o u l d now be done u s i n g t h e MABs. A l l o f t h e i s o l a t e s o f TMV r e c o v e r e d from s t o n e f r u i t s r esemble t h e tomato or Dahlemense i s o l a t e s ( 3 0 ) . The TMV i s o l a t e d from P. g l a n d u l o s a does not d i f f e r i n s e r o l o g i c a l o r p h y s i c a l c h a r a c t e r i s t i c s from t h e U l t y p e s t r a i n . As i n o t h e r f r u i t t r e e s , TMV p r o b a b l y o c c u r s i n P. g l a n d u l o s a as a l a t e n t i n f e c t i o n i n low c o n c e n t r a t i o n s . 75 R e f e r e n c e s C i t e d 1 . B a i l e y , J . M . , a n d D a v i d s o n , N . 1 9 7 6 . M e t h y l m e r c u r y a s a r e v e r s i b l e d e n a t u r i n g a g e n t f o r a g a r o s e g e l e l e c t r o p h o r e s i s . A n a l . B i o c h e m . 7 0 : 7 5 - 8 5 . 2 . B a r b a , M . , a n d C l a r k , M . F . 1 9 8 6 . D e t e c t i o n o f s t r a i n s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s b y F ( a b ) 2 - b a s e d i n d i r e c t E L I S A . A c t a H o r t i c . 1 9 3 : 2 9 7 - 3 0 4 . 3 . B a r - J o s e p h , M . , G a r n s e y , S . M . , a n d G o n s a l v e s , D . 1 9 7 9 . The c l o s t e r o v i r u s e s : a d i s t i n c t g r o u p o f e l o n g a t e d p l a n t v i r u s e s . A d v . V i r u s R e s . 2 5 : 9 3 - 1 6 8 . 4 . B a r - J o s e p h , M . , H u l l , R . , a n d L a n e , L . C . 1 9 7 4 . B i o p h y s i c a l a n d b i o c h e m i c a l c h a r a c t e r i z a t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s . V i r o l o g y 6 2 : 5 6 3 - 5 6 6 . 5 . B a r - J o s e p h , M . , a n d M u r a n t , A . F . 1 9 8 2 . C l o s t e r o v i r u s g r o u p . D e s c r i p t i o n s o f P l a n t V i r u s e s N o . 2 6 0 . C . M . I . / A . A . B . , Kew, S u r r e y , E n g l a n d . 3 p p . 6. B o l t o n , A . E . , a n d H u n t e r , W . M . 1 9 7 3 . T h e l a b e l l i n g o f p r o t e i n s t o h i g h s p e c i f i c r a d i o a c t i v i t i e s b y c o n j u g a t i o n t o a I - c o n t a i n i n g a c y l a t i n g a g e n t . A p p l i c a t i o n t o t h e r a d i o i m m u n o a s s a y . B i o c h e m . J . 1 3 3 : 5 2 9 - 5 3 9 . 7 . B u r g y a n , J . , B e c z n e r , L . , a n d N e m e t h , M . 1 9 8 0 . R e l a t i o n s h i p among some t o b a m o v i r u s e s I I . S e r o l o g i c a l c h a r a c t e r i z a t i o n o f a t o b a c c o m o s a i c v i r u s i s o l a t e d f r o m p l u m . A c t a H o r t i c . 9 4 : 3 3 9 - 3 4 8 . 8 . C a m p b e l l , A . I . , a n d B o u l d , C . 1 9 7 0 . V i r u s , f e r t i l i z e r a n d r o o t s t o c k e f f e c t s on t h e g r o w t h a n d p r e c o c i t y o f y o u n g a p p l e t r e e s . J . H o r t i c . S c i . 4 5 : 7 5 - 8 5 . 9 . C h a i r e z , R . , a n d L i s t e r , R . M . 1 9 7 3 . A c o m p a r i s o n o f two s t r a i n s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s . P h y t o p a t h o l o g y 6 3 : 1 4 5 8 - 1 4 6 4 . 1 0 . C l a r k , M . F . , a n d A d a m s , A . N . 1 9 7 7 . C h a r a c t e r i s t i c s o f t h e m i c r o p l a t e m e t h o d o f enzyme l i n k e d i m m u n o s o r b e n t a s s a y f o r t h e d e t e c t i o n o f p l a n t v i r u s e s . J . G e n . V i r o l . 3 4 : 4 7 5 - 4 8 3 . 1 1 . C o l i n , J . , a n d V e r h o y e n , M . 1 9 7 5 . I d e n t i f i c a t i o n d e s v i r u s c h e z l e s P r u n u s o r n a m e n t a u x e n B e l g i q u e . R e v . A g r i c . 4 : 9 3 5 -9 5 1 . 1 2 . C r o p l e y , R . 1 9 6 8 . C o m p a r i s o n o f some a p p l e l a t e n t v i r u s e s . A n n . A p p l . B i o l . 6 1 : 3 6 1 - 4 2 2 . 76 1 3 . C r o p l e y , R . 1 9 6 9 . A p p l e c h l o r o t i c l e a f s p o t ( s u p p l e m e n t ) . P a g e s 1 0 - 1 3 i n : V i r u s D i s e a s e s o f A p p l e s a n d P e a r s . T e c h . Commun. N o . 30 , S u p p l . N o s . 2 / 3 / 4 B u r . H o r t . , E . M a i l i n g . 1 4 . C r o p l e y , R . , W o l f s w i n k e l , L . D . , a n d P o s n e t t e , A . F . 1 9 6 3 . The i d e n t i f c a t i o n o f some v i r u s e s i n f e c t i n g a p p l e , p e a r , a n d q u i n c e . P h y t o p a t h o l . M e d i t . 2 : 1 3 2 - 1 3 6 . 1 5 . D a w s o n , W . O . , B u b r i c k , P . , a n d G r a n t h a m , G . L . 1 9 8 8 . M o d i f i c a t i o n s o f t h e t o b a c c o m o s a i c v i r u s c o a t p r o t e i n g e n e a f f e c t i n g r e p l i c a t i o n , m o v e m e n t , a n d s y m p t o m a t o l o g y . P h y t o p a t h o l o g y 7 8 : 1 8 3 - 7 8 9 . 1 6 . D a w s o n , W . O . , a n d D o d d s , J . A . 1 9 8 2 . C h a r a c t e r i z a t i o n o f s u b -g e n o m i c d o u b l e - s t r a n d e d RNAs f r o m v i r u s - i n f e c t e d p l a n t s . B i o c h e m . B i o p h y s . R e s . Commun. 1 0 7 : 1 2 3 0 - 1 2 3 5 . 1 7 . D e s v i g n e s , J . C . , a n d B o y e , R . 1 9 8 8 . D i f f e r e n t d i s e a s e s c a u s e d b y t h e c h l o r o t i c l e a f s p o t v i r u s on t h e f r u i t t r e e s . A c t a H o r t i c . 2 3 5 : 3 1 - 3 8 . 1 8 . D e t i e n n e , G . , D e l b o s , R . , a n d D u n e y , J . 1 9 8 0 . U s e a n d v e r s a t i l i t y o f t h e i m m u n o e n z y m a t i c E L I S A p r o c e d u r e i n t h e d e t e c t i o n o f d i f f e r e n t s t r a i n s o f an a p p l e c h l o r o t i c l e a f s p o t v i r u s . A c t a H o r t i c . 9 4 : 3 9 - 4 5 . 1 9 . D o d d s , J . A . , a n d B a r - J o s e p h , M . 1 9 8 3 . D o u b l e - s t r a n d e d RNA f r o m p l a n t s i n f e c t e d w i t h c l o s t e r o v i r u s e s . P h y t o p a t h o l o g y 7 3 : 4 1 9 - 4 2 3 . 2 0 . D u n e z , J . , D e l b o s , R . , a n d B e r t r a n e t , R . 1 9 7 3 . L e r 6 1 e de d i f f e r e n t s f a c t e u r s de s t a b i l i z a t i o n a p p l i q u e s a l a p u r i f i c a t i o n d u v i r u s d u c h l o r o t i c l e a f s p o t d u p o m m i e r ( C L S V ) . A n n . P h y t o p a t h o l . 5 : 2 5 5 - 2 6 4 . 2 1 . D u n e z , J . , M a r e n a u d , C , D e l b o s , R . , a n d L a n s a c , M . 1 9 7 2 . V a r i a b i l i t y o f symptoms i n d u c e d b y t h e a p p l e c h l o r o t i c l e a f s p o t (CLSV) - a t y p e o f C L S V p r o b a b l y r e s p o n s i b l e f o r b a r k s p l i t d i s e a s e o f p r u n e t r e e s . P l a n t D i s . R e p . 5 6 : 2 9 3 - 2 9 5 . 2 2 . D u n n , D . B . , a n d H i t c h b o r n , J . H . 1 9 6 5 . T h e u s e o f b e n t o n i t e i n t h e p u r i f i c a t i o n o f p l a n t v i r u s e s . V i r o l o g y 2 5 : 1 7 1 - 1 9 2 . 2 3 . E s a u , K . 1 9 6 8 . V i r u s e s i n H o s t P l a n t s . F o r m , D i s t r i b u t i o n , a n d P a t h o l o g i c a l E f f e c t s . U n i v . o f W i s c o n s i n P r e s s , M a d i s o n , W i s c o n s i n . 225 p p . 2 4 . F l e g g , C . L . , a n d C l a r k , M . F . 1 9 7 9 . The d e t e c t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s b y a m o d i f i e d p r o c e d u r e o f enzyme l i n k e d i m m u n o s o r b e n t a s s a y . A n n . A p p l . B i o l . 9 1 : 6 1 - 6 5 . 77 2 5 . F r a n c k i , R . I . B . , M i l n e , R . G . , a n d H a t t a , T . 1 9 8 5 . C l o s t e r o v i r u s g r o u p . P a g e s 2 1 9 - 2 3 4 i n : A t l a s o f P l a n t V i r u s e s , V o l u m e 2 . CRC P r e s s I n c . , B o c a R a t o n , F l o r i d a . 2 6 . F r i d l u n d , P . R . 1 9 8 2 . D i s t r i b u t i o n o f c h l o r o t i c l e a f s p o t v i r u s - i n f e c t e d b u d s o n v a r i o u s l e n g t h s o f a p p l e b u d s t i c k s i n s u c c e s s i v e y e a r s . A c t a H o r t i c . 1 3 0 : 8 5 - 8 8 . 2 7 . F u c h s , E . 1 9 8 0 . S e r o l o g i c a l d e t e c t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s (CLSV) a n d a p p l e s t e m g r o o v i n g v i r u s (SGV) i n a p p l e . A c t a H o r t i c . 9 4 : 6 9 - 7 3 . 2 8 . F u l t o n , R . W . 1 9 6 7 . P u r i f i c a t i o n a n d s e r o l o g y o f r o s e m o s a i c v i r u s . P h y t o p a t h o l o g y 5 7 : 1 1 9 7 - 1 2 0 1 . 2 9 . G i b b s , A . , a n d H a r r i s o n , B . 1 9 7 6 . P l a n t V i r o l o g y . T h e P r i n c i p l e s . E d w a r d A r n o l d L t d . , L o n d o n . 2 92 p p . 3 0 . G i l m e r , R . M . 1 9 6 7 . A p p l e c h l o r o t i c l e a f s p o t a n d t o b a c c o m o s a i c v i r u s e s i n c h e r r y . P l a n t D i s . R e p . 5 1 : 8 2 3 - 8 2 5 . 3 1 . G i l m e r , R . M . , a n d M i n k , G . I . 1 9 7 1 . L a t e n t v i r u s e s o f a p p l e I I I . I n d e x i n g w i t h C h e n o p o d i u m q u i n o a . N . Y . S t . A g r . E x p . S t a . , S e a r c h 1 : 1 5 - 2 1 . 3 2 . G o l d , A . H . 1 9 6 1 . A n t i h o s t s e r u m i m p r o v e s p l a n t v i r u s p u r i f i c a t i o n . P h y t o p a t h o l o g y 5 1 : 5 6 1 - 5 6 5 . 3 3 . G o n s a l v e s , D . , T r u j i l l o , E . , a n d H o c h , H . C . 1 9 8 6 . P u r i f i c a t i o n a n d some p r o p e r t i e s o f a v i r u s a s s o c i a t e d w i t h cardamom m o s a i c , a new member o f t h e p o t y v i r u s g r o u p . P l a n t D i s . 7 0 : 6 5 - 6 9 . 3 4 . G r i i n t z i g , M . , a n d F u c h s , E . 1 9 8 7 . [ C o m p a r i s o n o f d i f f e r e n t m e t h o d s f o r d e t e c t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s i n p l u m t r e e s . A r c h i v f u r P h y t o p a t h o l o g i e u n d P f l a n z e n s h u t z . 2 3 : 2 1 - 3 0 ] ( A b s t r . ) R e v . P l a n t P a t h o l . 6 7 : 1 . 3 5 . H a m i l t o n , R . I . , E d w a r d s o n , J . R . , F r a n c k i , R . I . B . , H s u , H . T . , H u l l , R . , K o e n i g , R . , a n d M i l n e , R . G . 1 9 8 1 . G u i d e l i n e s f o r t h e i d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f p l a n t v i r u s e s . J . G e n . V i r o l . 5 4 : 2 2 3 - 2 4 1 . 3 6 . H a n s e n , A . J . , a n d G r e e n , L . 1 9 8 3 . P o t e n t i a l o f r i b a v i r i n f o r t r e e f r u i t v i r u s i n h i b i t i o n . A c t a H o r t i c . 1 3 0 : 1 8 3 - 1 8 4 . 3 7 . H a n s e n , A . J . , a n d L a n e , W . D . 1 9 8 5 . E l i m i n a t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s f r o m a p p l e s h o o t c u l t u r e s b y r i b a v i r i n . P l a n t D i s . 6 9 : 1 3 4 - 1 3 5 . 78 3 8 . H o l m e s , F . O . 1 9 4 6 . A c o m p a r i s o n o f t h e e x p e r i m e n t a l h o s t r a n g e s o f t o b a c c o - e t c h a n d t o b a c c o - m o s a i c v i r u s e s . P h y t o p a t h o l o g y 3 6 : 6 4 3 - 6 5 9 . 3 9 . H s u , C . H . , S e h g a l , O . P . , a n d P i c k e t t , E . E . 1 9 7 6 . S t a b i l i z i n g e f f e c t o f d i v a l e n t m e t a l i o n s o n v i r i o n s o f s o u t h e r n b e a n m o s a i c . V i r o l o g y 6 9 : 5 8 7 - 5 9 5 . 4 0 . H u l l , R . 1 9 7 7 . T h e s t a b i l i z a t i o n o f t h e p a r t i c l e o f t u r n i p r o s e t t e v i r u s a n d o f o t h e r members o f t h e s o u t h e r n b e a n m o s a i c v i r u s g r o u p . V i r o l o g y 7 9 : 5 8 - 6 6 . 4 1 . J o h n s o n , D . A . , G a u t s c h , J . W . , S p o r t s m a n , J . R . , a n d E l d e r , J . H . 1 9 8 4 . I m p r o v e d t e c h n i q u e u t i l i z i n g n o n f a t d r y m i l k f o r a n a l y s i s o f p r o t e i n s a n d n u c l e i c a c i d s t r a n s f e r r e d t o n i t r o c e l l u l o s e . Gene A n a l . T e c h n . 1 : 3 - 8 . 4 2 . K a l a s h j a n , J . A . , a n d L i p a r t i a , Z . M . 1 9 8 6 . A p p l e c h l o r o t i c l e a f s p o t v i r u s (CLSV) i n a f f e c t e d t i s s u e s a n d t h e p o s s i b i l i t y o f i t s d e t e c t i o n b y immune e l e c t r o n m i c r o s c o p y ( I E M ) . A c t a H o r t i c . 1 9 3 : 3 1 1 - 3 1 7 . 4 3 . K a w a s h i m a , N . , a n d W i l d m a n , S . G . 1 9 7 0 . F r a c t i o n I p r o t e i n . A n n u . R e v . P l a n t P h y s i o l . 2 1 : 3 2 5 - 3 5 8 . 4 4 . K e r l a n , C , M i l l e , B . , a n d D u n e z , J . 1 9 8 1 . I m m u n o s o r b e n t e l e c t r o n m i c r o s c o p y f o r d e t e c t i n g a p p l e c h l o r o t i c l e a f s p o t a n d p l u m p o x v i r u s e s . P h y t o p a t h o l o g y 7 1 : 4 0 0 - 4 0 4 . 4 5 . K o h l e r , G . , a n d M i l s t e i n , C . 1 9 7 5 . C o n t i n u o u s c u l t u r e s o f f u s e d c e l l s s e c r e t i n g a n t i b o d y o f p r e d e f i n e d s p e c i f i c i t y . N a t u r e 2 5 6 : 4 9 5 - 4 9 7 . 4 6 . K u r p p a , A . , a n d M a r t i n , R . R . 1 9 8 6 . U s e o f d o u b l e - s t r a n d e d RNA f o r d e t e c t i o n a n d i d e n t i f i c a t i o n o f v i r u s d i s e a s e s i n R u b u s s p e c i e s . A c t a H o r t i c . 1 8 6 : 5 1 - 6 2 . 4 7 . L a e m m l i , U . K . 1 0 7 0 . C l e a v a g e o f s t r u c t u r a l p r o t e i n s d u r i n g t h e a s s e m b l y o f t h e h e a d o f b a c t e r i o p h a g e T 4 . N a t u r e 2 2 7 : 6 8 0 -6 8 5 . 4 8 . L a u l h e r e , J . P . , L e s c u r e , A . M . , a n d B r i a t , J . F . 1 9 8 8 . P u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f f e r r i t i n s f r o m m a i z e , p e a a n d s o y a b e a n s e e d s . J . B i o l . C h e m . 2 6 3 : 1 0 2 8 9 - 1 0 2 9 4 . 4 9 . L e g r a n d , G . , a n d V e r h o y e n , M . 1 9 8 5 . U s e o f a c o m p l e x s u c r o s e -p o l y e t h y l e n e g l y c o l g r a d i e n t f o r p u r i f y i n g a p p l e c h l o r o t i c l e a f s p o t v i r u s . P a r a s i t i c a 4 1 : 5 7 - 6 5 . 79 5 0 . L e m m e t t y , A . 1 9 8 8 . I s o l a t i o n a n d p u r i f i c a t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s a n d i t ' s o c c u r r e n c e i n F i n n i s h o r c h a r d s . A c t a H o r t i c . 2 3 5 : 1 7 7 - 1 8 0 . 5 1 . L i s t e r , R . M . , B a n c r o f t , J . B . , a n d N a d a k a v u k a r e n , M . J . 1 9 6 5 . Some s a p - t r a n s m i s s a b l e v i r u s e s f r o m a p p l e . P h y t o p a t h o l o g y 5 5 : 8 5 9 - 8 7 0 . 5 2 . L i s t e r , R . M . , a n d H a d i d i , A . F . 1 9 7 1 . Some p r o p e r t i e s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s a n d t h e i r r e l a t i o n t o p u r i f i c a t i o n p r o b l e m s . V i r o l o g y 4 5 : 2 4 6 - 2 5 1 . 5 3 . L l a c e r , G . , a n d C a m b r a , R . 1 9 7 5 . A n o m a l i e s d a n s l e c o m p o r t e m e n t d e s c e r t a i n e s v a r i e t e s d e s p e c h e r e t d ' a b r i c o t i e r g r e f f e s s u r d i v e r s t y p e s d u p r u n i e r e t l e u r p o s s i b l e r e l a t i o n a v e c l e v i r u s de c h l o r o t i c l e a f s p o t . A c t a H o r t i c . 4 4 : 9 9 - 1 0 6 . 5 4 . L u c k w i l l , L . C . , a n d C a m p b e l l , A . I . 1 9 5 9 . M a l u s p l a t y c a r p a as a n a p p l e v i r u s i n d i c a t o r . J . H o r t i c . S c i . 3 4 : 2 4 8 - 2 5 2 . 5 5 . M a r e n a u d , D . 1 9 6 8 . M i s e e n e v i d e n c e s u r l ' e s p e c e a b r i c o t i e r , d ' u n e e n c o m p a t i b i l i t i e i n t r a - s p e c i f i q u e d u e a l a p r e s e n c e d ' u n v i r u s d u t y p e c h l o r o t i c l e a f s p o t . A n n . E p i p h y t . 9 : 2 2 5 -2 4 5 . 5 6 . M a r e n a u d , C , D u n e z , J . , a n d B e r n h a r d , R . 1 9 7 6 . I d e n t i f i c a t i o n a n d c o m p a r i s o n o f d i f f e r e n t s t r a i n s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s a n d p o s s i b i l i t i e s o f c r o s s p r o t e c t i o n . A c t a H o r t i c . 6 7 : 2 1 9 - 2 2 6 . 5 7 . M a r t i n , R . R . 1 9 8 7 . T h e u s e o f m o n o c l o n a l a n t i b o d i e s f o r v i r u s d e t e c t i o n . C a n . J . P l a n t P a t h o l . 9 : 1 7 7 - 1 8 1 . 5 8 . M i n k , G . I . 1 9 8 9 . A p p l e c h l o r o t i c l e a f s p o t . P a g e s 8 -19 i n : V i r u s a n d V i r u s l i k e D i s e a s e s o f Pome F r u i t s a n d S i m u l a t i n g N o n i n f e c t i o u s D i s o r d e r s . P . R . F r i d l u n d , e d . W a s h i n g t o n S t a t e U n i v . , C o o p e r a t i v e E x t e n s i o n . P u l l m a n , W a s h i n g t o n . 5 9 . M i n k , G . I . , a n d S h a y , J . R . 1 9 5 9 . P r e l i m i n a r y e v a l u a t i o n o f some R u s s i a n a p p l e v a r i e t i e s as i n d i c a t o r s f o r a p p l e v i r u s e s . P l a n t D i s . R e p . ( S u p p l . ) 2 5 4 : 1 3 - 1 7 . 60 . N e m e t h , M . 1 9 8 6 . V i r u s , M y c o p l a s m a a n d R i c k e t t s i a D i s e a s e s o f F r u i t T r e e s . A k a d e m i a i K i a d 6 , B u d a p e s t . 841 p p . 6 1 . O h k i , S . T . , Y o s h i k a w a , N . , I n o u y e , N . , a n d I n o u y e , T . 1 9 8 9 . C o m p a r a t i v e e l e c t r o n m i c r o s c o p y o f C h e n o p o d i u m q u i n o a l e a v e s i n f e c t e d w i t h a p p l e c h l o r o t i c l e a f s p o t , a p p l e s t e m g r o o v i n g , o r c i t r u s t a t t e r l e a f v i r u s . A n n . P h y t o p a t h o l . S o c . J p n . 5 5 : 2 4 5 - 2 4 9 . 80 6 2 . O i , V . T . , a n d H e r z e n b e r g , L . A . 1 9 8 0 . I m m u n o g l o b u l i n - p r o d u c i n g h y b r i d c e l l l i n e s . P a g e s 3 5 1 - 3 7 2 i n : S e l e c t e d M e t h o d s i n C e l l u l a r I m m u n o l o g y . B . B . M i s h e l a n d S . M . S h i i g i , e d s . W . H . F r e e m a n . New Y o r k . 6 3 . P a u n o v i c , S . 1 9 8 8 . P r o p e r t i e s o f two a p p l e c h l o r o t i c l e a f s p o t i s o l a t e s . A c t a H o r t i c . 2 3 5 : 3 9 - 4 7 . 64 . P e n a - I g l e s i a s , A . , a n d A y u s o - G o n z a l e s , P . 1 9 7 5 . P r e l i m i n a r y i d e n t i f i c a t i o n o f t h e v i r u s S p a n i s h a p r i c o t p s e u d o p o x ( v i r u e l a ) a n d a p r i c o t m o s a i c d i s e a s e s . A c t a H o r t i c . 4 4 : 2 5 5 -2 6 2 . 6 5 . P o s n e t t e , A . F . , a n d C r o p l e y , R . 1 9 6 4 . N a t u r a l s p r e a d o f c h l o r o t i c l e a f s p o t v i r u s i n a p p l e a n d q u i n c e . A n n . R e p . E a s t M a i l i n g R e s . S t a . f o r 1 9 6 3 : 1 1 5 - 1 1 7 . 6 6 . P o u l , F . , a n d D u n e z , J . 1 9 8 9 . P r o d u c t i o n a n d u s e o f m o n o c l o n a l a n t i b o d i e s f o r t h e d e t e c t i o n o f a p p l e c h l o r o t i c l e a f s p o t v i r u s . J . V i r o l . M e t h . 2 5 : 1 5 3 - 1 6 6 . 6 7 . S a k s e n a , K . N . , a n d M i n k , G . I . 1 9 6 9 . B i o l o g i c a l a n d s e r o l o g i c a l d e t e c t i o n o f c h l o r o t i c l e a f s p o t v i r u s f r o m a p p l e b l o s s o m t i s s u e . P h y t o p a t h o l o g y 5 9 : 8 7 7 - 8 7 8 . 6 8 . S a k s e n a , K . N . , a n d M i n k , G . I . 1 9 6 9 . P r o p e r t i e s o f an i n h i b i t o r o f a p p l e c h l o r o t i c l e a f s p o t v i r u s f r o m C h e n o p o d i u m q u i n o a . P h y t o p a t h o l o g y 5 9 : 6 1 - 6 3 . 6 9 . S a k s e n a , K . N . , a n d M i n k , G . I . 1 9 6 9 . P u r i f i c a t i o n a n d p r o p e r t i e s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s . P h y t o p a t h o l o g y 5 9 : 8 4 - 8 8 . 7 0 . S a m u e l , G . 1 9 3 4 . The movement o f t o b a c c o m o s a i c v i r u s w i t h i n t h e p l a n t . A n n . A p p l . B i o l . 2 1 : 9 0 - 1 1 1 . 7 1 . S w e e t , J . B . 1 9 8 0 . H e d g e r o w h a w t h o r n ( C r a t a e g u s s p p . ) a n d b l a c k t h o r n ( P r u n u s s p i n o s a ) as h o s t s o f f r u i t t r e e v i r u s e s i n B r i t a i n . A n n . A p p l . B i o l . 9 4 : 8 3 - 9 0 . 7 2 . T h o m a s , J . 1 9 8 3 . The p a r t i c l e l e n g t h o f an i s o l a t e o f a p p l e c h l o r o t i c l e a f s p o t v i r u s f r o m P r u n u s d o m e s t i c a . P h y t o p a t h o l . Z . 1 0 6 : 2 3 3 - 2 3 8 . 7 3 . T h o m p s o n , D . A . , a n d S t a c e - S m i t h , R . 1 9 8 8 . I s o l a t i o n o f a c l o s t e r o v i r u s a n d a t o b a m o v i r u s f r o m d w a r f f l o w e r i n g a l m o n d . ( A b s t r . ) C a n J . P l a n t P a t h o l . 1 0 : 3 7 4 . 7 4 . V a n d e r M e e r , F . A . 1 9 7 5 . P l a n t s p e c i e s o u t s i d e t h e g e n u s M a l u s a s i n d i c a t o r s f o r l a t e n t v i r u s e s o f a p p l e . A c t a H o r t i c . 4 4 : 2 1 3 - 2 2 0 . 81 7 5 . V a n d e r M e e r , F . A . 1 9 8 6 . O b s e r v a t i o n s on t h e e t i o l o g y o f some v i r u s d i s e a s e o f a p p l e a n d p e a r . A c t a H o r t i c . 1 9 3 : 7 3 - 7 4 . 7 6 . V a n K a t w i j k , W. 1 9 8 9 . P e a r r i n g p a t t e r n m o s a i c . P a g e s 168-174 i n : V i r u s a n d V i r u s l i k e D i s e a s e s o f Pome F r u i t s a n d S i m u l a t i n g N o n i n f e c t i o u s D i s o r d e r s . P . R . F r i d l u n d , e d . W a s h i n g t o n S t a t e U n i v . , C o o p e r a t i v e E x t e n s i o n , P u l l m a n , W a s h i n g t o n . 7 7 . V a n R e g e n m o r t e l , M . H . V . 1 9 8 6 . The p o t e n t i a l f o r u s i n g m o n o c l o n a l a n t i b o d i e s i n t h e d e t e c t i o n o f p l a n t v i r u s e s . P a g e s 8 9 -101 i n : D e v e l o p m e n t s i n A p p l i e d B i o l o g y 1 . D e v e l o p m e n t s a n d A p p l i c a t i o n s i n V i r u s T e s t i n g . R . C . A . J o n e s a n d L . T o r r a n c e , e d s . A s s o c . A p p l . B i o l . , W e l l e s b o u r n e , U . K . 7 8 . V i n o g r a d , J . , a n d H e a r s t , J . E . 1 9 6 2 . E q u i l i b r i u m s e d i m e n t a t i o n o f m a c r o m o l e c u l e s a n d v i r u s e s i n a d e n s i t y g r a d i e n t . P r o g r . C h e m . O r g . N a t u r . P r o d . 2 0 : 3 7 3 - 4 2 2 . 7 9 . W e l s h , M . F . , a n d N y l a n d , G . 1 9 6 5 . E l i m i n a t i o n a n d s e p a r a t i o n o f v i r u s e s i n a p p l e c l o n e s b y e x p o s u r e t o d r y h e a t . C a n . J . P l a n t S c i . 4 5 : 4 4 3 - 4 5 4 . 8 0 . Y a n a s e , H . , N a k a t a n i , F . , M u n a k a t a , T . , a n d M a c h i d a , K . 1 9 8 6 . D e t e c t i o n o f d i f f e r e n t s t r a i n s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s a n d a p p l e s t e m g r o o v i n g v i r u s b y d i r e c t d o u b l e a n t i b o d y s a n d w i c h m e t h o d a n d m o d i f i e d i n d i r e c t E L I S A ( F ( a b ' ) 2 p r o c e d u r e . B u l l . F r u i t T r e e R e s . S t n . J p n . 1 3 : 6 9 - 8 1 . 8 1 . Y o s h i k a w a , N . , a n d T a k a h a s h i , T . 1 9 8 8 . P r o p e r t i e s o f RNAs a n d p r o t e i n s o f a p p l e s t e m g r o o v i n g a n d a p p l e c h l o r o t i c l e a f s p o t v i r u s e s . J . G e n . V i r o l . 6 9 : 2 4 1 - 2 4 5 . 8 2 . Z a g u l a , K . R . , A r e f , N . M . , a n d R a m s d e l l , D . C . 1 9 8 9 . P u r i f i c a t i o n , s e r o l o g y , a n d some p r o p e r t i e s o f a m e c h a n i c a l l y t r a n s m i s s i b l e v i r u s a s s o c i a t e d w i t h g r e e n r i n g m o t t l e d i s e a s e i n p e a c h a n d c h e r r y . P h y t o p a t h o l o g y 7 9 : 4 5 1 - 4 5 6 . 8 3 . Z a i t l i n , M . , a n d I s r a e l , H . W . 1 9 7 5 . T o b a c c o m o s a i c v i r u s ( t y p e s t r a i n ) . D e s c r i p t i o n s o f P l a n t V i r u s e s N o . 1 5 1 . C M . I . / A . A . B . Kew, S u r r e y , E n g l a n d . 5 p p . 84 . Z e e , F . , G o n s a l v e s , D . , G o h e e n , A . , K i m , K . S . , P o o l , R . , a n d L e e , R . F . 1 9 8 7 . C y t o p a t h o l o g y o f l e a f r o l l - d i s e a s e d g r a p e v i n e s a n d t h e p u r i f i c a t i o n a n d s e r o l o g y o f a s s o c i a t e d c l o s t e r o v i r u s l i k e p a r t i c l e s . P h y t o p a t h o l o g y 7 7 : 1 4 2 7 - 1 4 3 4 . 

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