Rev. sci. tech. Off. int. Epiz., 1995,14 (3), 811-818

Reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections T. STADEJEK*, Z. PEJSAK*, M. KWINKOWSKI**, A. OKRUSZEK** and S. WINIARCZYK ***

Summary: An assay based on reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection of hog cholera virus (HCV) and bovine virus diarrhoea virus (BVDV) in cell culture. In this study, a precipitate of the supernatants derived from cell cultures infected with HCV and BVDV was used in RT reactions, in place of extracted viral RNA. Both RT and PCR were performed using recombinant T h e r m u s t h e r m o p h i l u s ( r T t h ) DNA polymerase. The specificity of the RT-PCR products was confirmed by hybridisation with a digoxygenin-labelled DNA probe. The results not only show that the stage of RNA isolation can be bypassed, but also illustrate an easy and efficient means of obtaining templates suitable for identification and characterisation of HCV and BVDV in tissue culture by RTPCR. K E Y W O R D S : B o v i n e virus d i a r r h o e a virus - H o g cholera virus - Pestivirus P o l y m e r a s e chain reaction - R e v e r s e transcription - rTth D N A p o l y m e r a s e .

INTRODUCTION T h e g e n u s Pestivirus h a s b e e n classified in t h e family F l a v i v i r i d a e ( 2 0 ) , a n d v i r u s e s b e l o n g i n g t o t h i s g e n u s a r e r e c o g n i s e d a s b e i n g of g r e a t e p i z o o t i c a n d e c o n o m i c importance. T h e genus comprises hog cholera virus ( H C V ) , bovine virus d i a r r h o e a virus ( B V D V ) a n d B o r d e r d i s e a s e v i r u s ( B D V ) , w h i c h a r e c l o s e l y r e l a t e d b o t h antigenically a n d s t r u c t u r a l l y . T h e s e v i r u s e s a r e s m a l l a n d e n v e l o p e d , a n d t h e d i a m e t e r of t h e v i r i o n r a n g e s f r o m 40 n m t o 50 n m (12). T h e g e n o m e c o n s i s t s of a s i n g l e - s t r a n d e d , n o n - p o l y a d e n y l a t e d , p o s i t i v e l y - p o l a r i s e d r i b o n u c l e i c acid ( R N A ) ( 6 ) , a n d h a s a size of 12,280 n u c l e o t i d e s ( H C V , B r e s c i a s t r a i n ) o r 1 2 , 5 7 3 n u c l e o t i d e s ( B V D V , N A D L [ N a t i o n a l A n i m a l D i s e a s e L a b o r a t o r y , A m e s , I o w a ] s t r a i n ) ( 3 , 1 3 ) . T h e R N A of pestivirus c o d e s for a p p r o x i m a t e l y 4,000 a m i n o acids (4, 11).

* Department of Swine Diseases, National Veterinary Research Institute, 57 Partyzantow St., 24-100 Pulawy, Poland. ** Department of Bioorganic Chemistry, Center for Molecular and Macromolecular Studies, Polish Academy of Sciences, 112 Sienkiewicz St., 90-363 Lodz, Poland. *** Department of Epizootiology, Veterinary Faculty, Agricultural University, 30 Gleboka St., 20-612 Lublin, Poland.

812 H C V - t h e c a u s a t i v e v i r u s of h o g c h o l e r a ( c l a s s i c a l s w i n e f e v e r ) , o n e of t h e m o s t d a n g e r o u s i n f e c t i o u s d i s e a s e s of s w i n e - is c o n s i d e r e d t h e m o s t i m p o r t a n t of t h e p e s t i v i r u s e s , d u e t o t h e e c o n o m i c losses c a u s e d t o t h e pig i n d u s t r y b y this d i s e a s e . T h e a b i l i t y of p e s t i v i r u s e s t o c r o s s s p e c i e s b a r r i e r s ( 7 ) , t h e i r c l o s e r e l a t i o n s h i p , a n d t h e f r e q u e n t o c c u r r e n c e of a t y p i c a l f o r m s of i n f e c t i o n c a u s e d b y H C V s t r a i n s w i t h m i l d or low v i r u l e n c e (12) c o m p l i c a t e t h e d i a g n o s i s a n d m a k e c o n t r o l of t h e d i s e a s e difficult. In r e c e n t years, significant p r o g r e s s h a s b e e n m a d e in r e s e a r c h o n pestiviruses. C o m p l e t e n u c l e o t i d e s e q u e n c e d a t a h a v e r e c e n t l y b e e n p u b l i s h e d for t h e N A D L , O s l o s s a n d SD1 s t r a i n s of B V D V , a n d t h e A l f o r t a n d B r e s c i a s t r a i n s of H C V ( 3 , 5 , 1 1 , 1 3 , 1 5 ) ; t h e s e h a v e e n a b l e d s t u d i e s t o b e c o n d u c t e d o n n e w d i a g n o s t i c m e t h o d s at the g e n e t i c l e v e l , i n c l u d i n g t h e p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) ( 1 6 ) . T h i s m e t h o d uses r e p e a t e d cycles of r e a c t i o n of t a r g e t D N A w i t h a p p r o p r i a t e o l i g o n u c l e o t i d e p r i m e r s and t h e r m o s t a b l e D N A p o l y m e r a s e . T w e n t y t o t h i r t y P C R cycles c a n p r o d u c e a n i n c r e a s e of at least 1 0 in t h e a m o u n t of a r e q u i r e d s e q u e n c e . T h e p e s t i v i r u s g e n o m e is c o n s t r u c t e d of R N A , a n d t h e P C R m u s t t h e r e f o r e b e p r e c e d e d b y a r e v e r s e t r a n s c r i p t i o n ( R T ) reaction, to transcribe genetic information from R N A o n t o the c o m p l e m e n t a r y D N A . S e v e r a l i n v e s t i g a t o r s h a v e r e p o r t e d P C R - b a s e d d i a g n o s t i c t e s t s for B V D V a n d H C V ( 1 , 8, 9, 10, 18, 19). T h i s r e p o r t d e s c r i b e s t h e a p p l i c a t i o n of R T - P C R for t h e d e t e c t i o n of p e s t i v i r u s e s in s u p e r n a t a n t s of cell-line c u l t u r e s . 5

MATERIAL AND METHODS Virus strains T h e following virus strains w e r e u s e d : -

t e n field isolates of H C V from o u t b r e a k s in P o l a n d

-

t h e B r e s c i a strain of H C V t h e N A D L a n d O r e g o n strains of B V D V .

H C V s t r a i n s w e r e g r o w n for 7 2 h o n p i g k i d n e y ( P K ) 1 5 c e l l s . B V D V s t r a i n s w e r e g r o w n o n M a d i n - D a r b y b o v i n e k i d n e y ( M D B K ) c e l l s , u n t i l t h e c y t o p a t h i c effect reached 80-90%. Viral culture precipitation V i r a l c u l t u r e s w e r e f r o z e n a n d t h a w e d t h r e e t i m e s , t h e n c e n t r i f u g e d (15,000 x g for 10 m i n a t 4 ° C ) , a n d s u p e r n a t a n t s w e r e c o l l e c t e d . V i r a l p a r t i c l e s w e r e p r e c i p i t a t e d o v e r n i g h t w i t h 7 % p o l y e t h y l e n e glycol 20,000 at 4 ° C , c e n t r i f u g e d (15,000 x g for 1 h at 4 ° C ) a n d s u s p e n d e d in d o u b l e - d i s t i l l e d water. S u s p e n s i o n s w e r e s t o r e d a t - 2 0 ° C (21). R N A isolation R N A was isolated by the modified C h o m c z y n s k i m e t h o d (2). Viral suspension (150 µl) w a s s h a k e n with 400 µl of 6 M g u a n i d i n i u m i s o t h i o c y a n a t e a n d left for 10 min at r o o m t e m p e r a t u r e . T h e n 220 µl of 5 M N a C l w e r e a d d e d , f o l l o w e d b y 10 m i n incubation o n i c e . A f t e r e x t r a c t i o n w i t h 3 0 0 µl c h l o r o f o r m a n d c e n t r i f u g a t i o n ( 1 4 , 0 0 0 x g for 15 m i n ) , 600 µl of u p p e r p h a s e ( w a t e r ) w e r e t r a n s f e r r e d t o t h e n e w t u b e , a n d R N A was p r e c i p i t a t e d o v e r n i g h t w i t h 3 6 0 µl of 9 6 % e t h a n o l a t - 2 0 ° C . R N A w a s c o l l e c t e d by c e n t r i f u g a t i o n ( 1 4 , 0 0 0 x g for 4 5 m i n ) , r i n s e d c a r e f u l l y w i t h 7 0 % e t h a n o l , d r i e d and s u s p e n d e d in d o u b l e - d i s t i l l e d water. S u s p e n s i o n s w e r e s t o r e d at - 7 0 ° C .

813 Primers P r i m e r s w e r e s y n t h e s i s e d using t h e p h o s p h o r o a m i d i t e m e t h o d , f o l l o w e d b y t w o h i g h pressure liquid c h r o m a t o g r a p h y purification steps. P r i m e r s w e r e designed by B o y e et al. (1), and t h e i r p o s i t i o n s a n d s e q u e n c e s w e r e as follows: -

primer P1: 9 8 - ( 5 ' - G A G G C T A G C C A T G C C C T T A G T - 3 ' ) - 1 1 9

-

primer P 3 : 3 7 1 - ( 5 ' - T C A A C T C C A T G T G C C A T G T A C A G C A - 3 ' ) - 3 9 5 .

B o t h p r i m e r s w e r e d e s i g n e d t o flank t h e r e g i o n c o n t a i n i n g t h e j u n c t i o n b e t w e e n n o n coding a n d c o d i n g s e q u e n c e s a t t h e 5 ' e n d of t h e g e n o m e of t h e N A D L s t r a i n of B V D V , one of t h e m o s t c o n s e r v e d r e g i o n s in all p e s t i v i r u s e s . Reverse transcription Reverse transcription was performed using thermostable enzyme recombinant Thermus thermophilus (rTth) D N A p o l y m e r a s e ( 1 4 ) . T h e a c t i v i t y of t h i s e n z y m e depends o n t h e p r e s e n c e of m e t a l i o n s . I n t h e p r e s e n c e of m a g n e s i u m i o n s t h e e n z y m e has D N A - d e p e n d e n t D N A p o l y m e r a s e a c t i v i t y , w h e r e a s w i t h m a n g a n e s e i o n s it exhibits R N A - d e p e n d e n t D N A p o l y m e r a s e ( r e v e r s e t r a n s c r i p t a s e ) a c t i v i t y . T h e e n z y m a t i c r e a c t i o n w a s p e r f o r m e d in a 0.65 m l t u b e b y m i x i n g t o g e t h e r 2 µl of 10x reverse t r a n s c r i p t a s e b u f f e r ( 1 0 0 m M t r i s - H C 1 , p H 8.3; 900 m M K C l ) , 2 µl of 10 m M M n C l , 0.5 µl e a c h of 10 m M s o l u t i o n s of d A T P , d G T P , d C T P a n d d T T P , 2 µl of 15 µM aqueous s o l u t i o n of p r i m e r P 3 , a n d 2.5 u n i t s of rTth D N A p o l y m e r a s e . T o this s o l u t i o n were a d d e d e i t h e r 5 µl of R N A t e m p l a t e ( i s o l a t e d a s a b o v e ) o r 2 µl of c r u d e v i r a l precipitate. T h e r e a c t i o n m i x t u r e w a s t o p p e d u p w i t h w a t e r t o 20 µl. A f t e r i n c u b a t i o n of the m i x t u r e for 10 m i n at 7 0 ° C u n d e r a l a y e r of m i n e r a l oil, t h e r e a c t i o n w a s s t o p p e d b y transferring t h e t u b e t o a n ice b a t h . 2

Polymerase chain reaction P C R w a s p e r f o r m e d in t h e s a m e t u b e after a d d i t i o n of 80 µl of a m i x t u r e c o n t a i n i n g 64 µl of w a t e r , 8 µl of 10x c h e l a t i n g b u f f e r ( 5 0 % g l y c e r o l , 1 0 0 m M K C l , 7.5 m M e t h y l e n e g l y c o l t e t r a a c e t i c a c i d , 0 . 5 % T w e e n 2 0 ) , 6 µl of 2 5 m M M g C l a n d 2 µl of P1 p r i m e r s o l u t i o n ( 1 5 µ M ) . T h e f i n a l v o l u m e of t h e r e a c t i o n m i x t u r e w a s 100 µl. Amplification w a s p e r f o r m e d in 35 cycles of t h e following t h r e e s t e p s : d e n a t u r a t i o n s t e p for 45 sec at 9 4 ° C ; a n n e a l i n g s t e p for 1 m i n a t 6 0 ° C ; e x t e n s i o n s t e p for 1 m i n a t 7 2 ° C . T h e d e n a t u r a t i o n s t e p in t h e first cycle w a s p r o l o n g e d t o 2 m i n . T h e e x t e n s i o n s t e p in t h e last cycle w a s p r o l o n g e d t o 7 m i n . 2

Analysis of P C R products P C R p r o d u c t s w e r e directly a n a l y s e d b y a g a r o s e gel e l e c t r o p h o r e s i s ( 2 % a g a r o s e gel in T r i s - b o r a t e - e t h y l e n e d i a m i n e t e t r a a c e t i c acid [ T B E ] buffer for 30 m i n a t 5 V / c m ) w i t h a 100 b p D N A l a d d e r a s a m a r k e r . T h e D N A w a s 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 solution a n d visualised u n d e r 300 n m u l t r a - v i o l e t ( U V ) light (17). T h e specificity of t h e a m p l i f i e d p r o d u c t s w a s v e r i f i e d b y d o t b l o t h y b r i d i s a t i o n of t h e P C R p r o d u c t s immobilised on a m e m b r a n e , with a digoxygenin-labelled p r o b e . T h e p r o b e was p r e p a r e d b y r a n d o m p r i m e r l a b e l l i n g of t h e D N A of p E M B L 1 8 ( + ) p l a s m i d c o n t a i n i n g the D N A c o m p l e m e n t a r y t o R N A of H C V A l f o r t s t r a i n , f r o m t h e 5 ' e n d (11). L a b e l l i n g was p e r f o r m e d u s i n g a d i g o x y g e n i n D N A l a b e l l i n g kit, e x a c t l y in a c c o r d a n c e w i t h t h e instructions of t h e m a n u f a c t u r e r . H y b r i d i s a t i o n w a s p e r f o r m e d as d e s c r i b e d below. P C R p r o d u c t s w e r e d e n a t u r e d a t 9 8 ° C f o r 10 m i n , t h e n r a p i d l y c o o l e d in a n i c e b a t h a n d loaded i n t o t h e m e m b r a n e . D N A w a s t h e n i m m o b i l i s e d on t h e m e m b r a n e by U V irradiation a n d h y b r i d i s e d w i t h a p r o b e . A l l p r o c e d u r e s ( p r e h y b r i d i s a t i o n , h y b r i d i s a t i o n and v i s u a l i s a t i o n of h y b r i d i s e d d o t s ) w e r e p e r f o r m e d u s i n g a d i g o x y g e n i n D N A detection kit, exactly in a c c o r d a n c e w i t h t h e i n s t r u c t i o n s of t h e m a n u f a c t u r e r .

814

RESULTS T h e t i t r e s of t e n field i s o l a t e s p r o p a g a t e d o n P K 15 cells d i f f e r e d significantly from each other and ranged between 1 0 and 1 0 T C I D (50 % t i s s u e c u l t u r e infective d o s e ) p e r m l . E q u a l v o l u m e s ( 2 5 m l ) of c e l l - c u l t u r e m e d i u m of e a c h i s o l a t e w e r e p r o c e s s e d a n d u s e d for R T - P C R . 3 . 5

7 . 8

5 0

E n z y m a t i c a m p l i f i c a t i o n of b o t h t h e R N A e x t r a c t s a n d c r u d e viral p r e c i p i t a t e s of ten field i s o l a t e s , f o l l o w e d b y a n a l y s i s of R T - P C R p r o d u c t s b y a g a r o s e gel e l e c t r o p h o r e s i s a n d e t h i d i u m b r o m i d e s t a i n i n g , r e v e a l e d ( i n b o t h i n s t a n c e s ) b a n d s a t t h e p o s i t i o n of f r a g m e n t s of t h e p r e d i c t e d s i z e , i . e . 3 0 0 b p . V a r i a t i o n s i n t h e i n t e n s i t y of b a n d s in e t h i d i u m b r o m i d e - s t a i n e d gels b e t w e e n t h e a m p l i f i e d f r a g m e n t s d i d n o t c o r r e l a t e with t h e t i t r e of t h e virus strain; t h e e x a c t r e a s o n for this p h e n o m e n o n is n o t clear. T o define t h e s e n s i t i v i t y of t h e R T - P C R r e a c t i o n , t e n - f o l d s e r i a l d i l u t i o n s of t h e t i t r a t e d H C V B r e s c i a s t r a i n w e r e p e r f o r m e d a n d p r o c e s s e d as a b o v e . T h e l o w e s t d e t e c t a b l e a m o u n t of virus w a s 1 T C I D . C o n t r o l e x p e r i m e n t s , p e r f o r m e d o n P K 1 5 a n d M D B K cell lines n o t i n f e c t e d w i t h v i r u s e s , d i d n o t r e s u l t in s p e c i f i c a m p l i f i c a t i o n . N o v i s i b l e p r o d u c t b a n d s w e r e o b s e r v e d i n a n a g a r o s e gel a f t e r e l e c t r o p h o r e s i s of t h e R T - P C R p r o d u c t (Fig. l a ) . T h e r e w a s n o h y b r i d i s a t i o n w i t h t h e D N A p r o b e , a l t h o u g h t h e digoxygeninl a b e l l e d D N A p r o b e h y b r i d i s e d t o all t h e R T - P C R p r o d u c t s o b t a i n e d w i t h R N A e x t r a c t s o r c r u d e p r e c i p i t a t e s , t h u s c o n f i r m i n g t h e specificity of t h e assay (Fig. l b ) . 5 0

Lane Lane Lane Lane Lane Lane Lane Lane Lane

1:100 bp ladder 2: HCV Brescia strain 3: HCV isolate 92371 4: HCV isolate 91748 5: HCV isolate 91749 6: negative control (uninfected PK15 cell line) 7: BVDV Oregon strain 8: BVDV NADL strain 9: negative control (uninfected MDBK cell line)

HCV: BVDV: PK: NADL:

hog cholera virus bovine virus diarrhoea virus pig kidney National Animal Disease Laboratory, Ames, Iowa MDBK: Madin-Darby bovine kidney

FiG.l Electrophoresis and hybridisation o f p o l y m e r a s e chain reaction amplification products derived from pestivirus-infected cell-culture precipitates a) agarose gel (2%) electrophoresis b) dot blot hybridisation with a digoxygenin-labelled D N A p r o b e

815 T h e s t a b i l i t y of v i r a l g e n o m e s w a s e v a l u a t e d d u r i n g l o n g - t e r m s t o r a g e in f r e e z e r s . While t h e a u t h o r s w e r e a b l e t o amplify s a m p l e s of p r e c i p i t a t e d v i r i o n s s t o r e d a t – 2 0 ° C within o n e year, t h e y c o u l d n o t amplify p u r i f i e d R N A s t o r e d a t - 7 0 ° C for o n e m o n t h .

CONCLUSION T h e m a j o r a d v a n t a g e of t h e m e t h o d d e s c r i b e d a b o v e is t h e a p p l i c a t i o n of t h e c r u d e viral p r e c i p i t a t e as a s u b s t r a t e for R T - P C R r e a c t i o n . T h e R N A of t h e virus c o n t a i n e d in the p r e c i p i t a t e is c a p s i d a t e d a n d is t h u s b e t t e r p r o t e c t e d a g a i n s t e n z y m a t i c a n d c h e m i c a l d e g r a d a t i o n d u r i n g h a n d l i n g a n d p r o l o n g e d s t o r a g e . T h e s t a b i l i t y of t h e p r e c i p i t a t e d viral p r e p a r a t i o n s e n a b l e s t h e a n a l y s e d s a m p l e s t o b e s t o r e d for a l o n g t i m e a n d t h e n used w h e n n e c e s s a r y , e.g. in c o m p a r a t i v e s t u d i e s . T h e a p p l i c a t i o n of t h e r T t h D N A p o l y m e r a s e - w h i c h is a l s o a b l e t o r e v e r s e transcribe R N A t e m p l a t e s - simplified t h e R T - P C R p r o c e d u r e , as b o t h r e a c t i o n s w e r e performed in o n e t u b e w i t h o u t c u m b e r s o m e m a n i p u l a t i o n s . T h e high t e m p e r a t u r e (70°C) r e q u i r e d for rTth D N A p o l y m e r a s e a c t i v i t y e n h a n c e d t h e d e n a t u r a t i o n of t h e viral p r o t e i n s a n d R N A , p r o m o t i n g m o r e efficient s y n t h e s i s of c o m p l e m e n t a r y D N A through t h e s e c o n d a r y R N A s t r u c t u r e of p e s t i v i r u s R N A (11). A n a d d i t i o n a l a d v a n t a g e of p e r f o r m i n g R T a t e l e v a t e d t e m p e r a t u r e is t h e i n h i b i t i o n of n u c l e a s e activity. It s h o u l d b e e m p h a s i s e d t h a t t h e c r u d e viral p r e c i p i t a t e , a l t h o u g h p r e p a r e d in a v e r y easy a n d s i m p l e way, a p p e a r e d t o b e a n a d e q u a t e t e m p l a t e for e n z y m a t i c a m p l i f i c a t i o n and c o m p l e t e l y b y p a s s e d t h e n e e d for R N A e x t r a c t i o n . F u r t h e r s t u d i e s a r e n e c e s s a r y t o investigate w h e t h e r a similar a p p r o a c h w o u l d b e useful in p r e p a r i n g s u i t a b l e t e m p l a t e s from o r g a n s of s l a u g h t e r e d a n i m a l s . A t p r e s e n t , t h i s a p p r o a c h s e e m s a p p l i c a b l e t o analytical s t u d i e s in v e t e r i n a r y virology.

ACKNOWLEDGEMENTS T h i s w o r k w a s c o n d u c t e d as p a r t of P r o j e c t N o . P L - A R S - 1 5 6 , in c o - o p e r a t i o n w i t h the N a t i o n a l A n i m a l D i s e a s e s C e n t e r in A m e s , I o w a ( U n i t e d S t a t e s of A m e r i c a ) a n d was p a r t l y f u n d e d b y t h e P o l i s h / A m e r i c a n M a r i a S k l o d o w s k a C u r i e J o i n t F u n d . T h e a u t h o r s a r e grateful t o D r H . - J . T h i e l of t h e F e d e r a l R e s e a r c h C e n t r e for V i r u s Diseases of A n i m a l s i n T ü b i n g e n ( G e r m a n y ) for p r o v i d i n g t h e H C V c l o n e 4.0 u s e d in the p r e p a r a t i o n of t h e p r o b e for h y b r i d i s a t i o n .

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816 LA TRANSCRIPTION RÉVERSE ASSOCIÉE À LA TECHNIQUE D'AMPLIFICATION E N C H A Î N E P A R P O L Y M É R A S E : U N E M É T H O D E DE D É T E C T I O N D E S P E S T I V I R O S E S . - T. S t a d e j e k , Z . Pejsak, M. Kwinkowski, A . Okruszek et S. Winiarczyk. Résumé : Une épreuve alliant la transcription réverse (reverse t r a n s c r i p t i o n : RT) à la technique d'amplification en chaîne par polymérase (polymerase chain reaction : PCR) a été appliquée à la détection des virus de la peste porcine classique et de la diarrhée virale bovine ( b o v i n e virus d i a r r h o e a : BVD) en culture cellulaire. Cette étude a utilisé un précipité réalisé à partir du surnageant des cultures infectées par les virus de la peste porcine classique et de la BVD dans des réactions de RT au lieu de l'acide ribonucléique (ARN) viral obtenu par extraction. Les techniques RT et PCR font toutes deux appel, dans leur application, à l'ADN polymérase recombiné de T h e r m u s t h e r m o p h i l u s (rTth). La spécificité des produits RT-PCR a été confirmée par hybridation à l'aide d'une sonde nucléique marquée à la digoxygénine. Les résultats montrent non seulement que le stade de l'isolement de TARN peut être dépassé, mais aussi que l'on peut obtenir, de manière simple et efficace, des modèles adaptés à l'identification et au typage des virus de la peste porcine classique et de la BVD en culture cellulaire grâce à la méthode RT-PCR. M O T S - C L É S : A D N p o l y m é r a s e xTth - A m p l i f i c a t i o n en c h a î n e p a r p o l y m é r a s e - Pestivirus - Transcription r é v e r s e - Virus d e la d i a r r h é e virale bovine - Virus de la peste porcine classique.

* * T R A N S C R I P C I Ó N I N V E R S A C O M B I N A D A C O N R E A C C I Ó N E N C A D E N A DE LA POLIMERASA COMO M É T O D O D E D E T E C C I Ó N D E INFECCIONES P E S T I V I R A L E S . - T. Stadejek, Z. Pejsak, M. Kwinkowski, A . Okruszek y S. Winiarczyk. Resumen: Los autores describen un ensayo para la detección del virus de la peste porcina clásica (hog c h o l e r a virus: HCV) y del virus de la diarrea viral bovina ( b o v i n e virus d i a r r h o e a v i r u s : BVDV) en cultivos celulares. Este método se basa en el acoplamiento entre la transcripción inversa ( r e v e r s e transcription: RT) y la reacción en cadena de la polimerasa (polymerase chain r e a c t i o n : PCR) (RT-PCR). Para las reacciones de RT, y en lugar de la extracción de ARN vírico, en este estudio se empleó un precipitado de los sobrenadantes obtenidos a partir de cultivos celulares infectados con HCV y BVDV. Tanto para la RT como para la PCR se utilizó ADN polimerasa recombinante de T h e r m u s t h e r m o p h i l u s (rTth). La especificidad de los productos de la RT-PCR se confirmó por hibridación con una sonda de ADN marcada con digoxigenina. Los resultados no sólo demuestran que, gracias al empleo asociado de las técnicas de RT-PCR, es posible obviar el paso del aislamiento del ARN, sino que ilustran también un procedimiento fácil y eficaz para obtener moldes adecuados para la identificación y caracterización por RT-PCR del HCV y del BVDV en cultivos tisulares. P A L A B R A S C L A V E : A D N rTth p o l i m e r a s a - P e s t i v i r u s - R e a c c i ó n e n c a d e n a d e la p o l i m e r a s a - T r a n s c r i p c i ó n i n v e r s a - V i r u s d e la d i a r r e a viral bovina - Virus de la peste porcina clásica.

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