PCR Seah Chow

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Arch Virol (1993) I33:157-170_ A r c h i v e s _Vi?ology Spr inger -Ver lag 1993P r i n t e d i n A u s t r i a

U s e o f N S 3 c o n s e n s u s p r im e r s f or 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 na m p l i f i c a t i o n a n d s e q u e n c i n g o f d e n g u e v i r u s e s

and o ther f lav iv i rusesV. T. K . C how, Catherine L. K . Sea h, and Y. C. Chan

Departm ent o f M icrobiology, Faculty o f Med icine, National University of S ingapore,Kent Ridge, SingaporeAccepted November 24, 1992

Summary. C on se nsus p r ime rs fo r the p o lyme ra se c ha in re a c t ion we re de s igne db a s e d o n c o n s e r v ed m o t i fs w i t h i n t he s e r in e p ro t e a s e a n d R N A h e li ca s e d o m a i n se nc ode d by the N S 3 gene s o f de ngue a n d o the r f l av iv iruse s . Ta rg e t f r a gm e n t so f 4 70 b p w e r e a m p l i fi e d o n c D N A t e m p l a t e s sy n t he s iz e d f r o m R N A s o f d e n g u etypes 1 , 2 , 3 , an d 4 , Japa nes e encep ha l i t i s , K un j in , a nd ye l low fever v i rusesu s i n g r a n d o m o r s p e ci fi c d o w n s t r e a m p r im e r s . P C R o f o l i g o (d T ) - p r i m e d c D N A sf r o m J a p a n e s e e n c e p h a l it i s a n d K u n j i n v i r a l R N A s d i d n o t y i el d t a r g e t b a n d s .As fe w a s 103 c op ie s o f de ngue v i ra l R NA c ou ld be de te c t e d . D i re c t DNAs e q u e n ci n g o f P C R p r o d u c t s o f r e fe r en c e s tr a in s o f d e n g u e 2 ( N G C ) , K u n j i n(M R M 61 C ) a nd ye l low fe ve r (17 D) v i ruse s de mon s t ra t e d c om ple te c onc ur re n c ewi th pub l i she d da ta . How e ve r , 2 nuc le o t ide di f fe re nc es we re obse rve d be twe e nour da ta fo r de ngue 3 H 87 s t r a in a nd the pub l i she d se que nc e , r e su l t ing in as ingle amino ac id d ispar i ty . Diffe rences a t 21 , 16 , and 11 nuc leot ide posi t ionswe re no te d be twe e n de ngue 1 Ha w a i i a nd S 275 /90 ; de ngue 4 H 241 a nd 814669;J a p a n e s e e n c e p h a l it i s N a k a y a m a a n d J a O A r S 9 82 v i r al s t r ai n s , c u l m i n a t i n g i non ly 4 , 1 a nd 1 a m ino a c id re s idue d i f fere nc e s, r espe c tive ly . The se a m ino a c idd i spa r i t i e s oc c u r re d ou t s ide pu ta t ive a c t ive s i t e s o f the e nz yma t i c doma ins ,e m p h a s i z i n g t h e i m p o r t a n t r o l e o f t h e N S 3 p r o t e i n i n f l av i v ir a l r e p l ic a t io n .T h i s R N A - P C R c o n s e n su s p r i m e r s t r at e g y c o u p l e d w i t h D N A s e q u e n ci n g r ep -re se n ts a va lua b le too l fo r the mole c u la r d i a gnos i s a nd e p ide m io logy o f de nguea nd o the r f l a v iv i ra l i n fe c t ions .

IntroductionO u t b r e a k s o f d e n g u e v i ru s i n f e c t i o n a n d t h e a s s o c i a te d s y n d r o m e s o f d e n g u efe ver , de ngue h a e m orrha g ic fe ver a nd the po te n t i a l ly f a t a l de ngue shoc k syn -d r o m e c o n s t i t u t e m a j o r p u b l i c h e a l t h p r o b l e m s i n t r o p i c a l a n d s u b - t r o p i c a l


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158 V .T .K . Chow e ta l.r e g ions a l l ove r the w or ld , inc lud ing the As ia - Pa c i f i c whe r e the A e d e s m o s q u i t ove c to r i s p r e va le n t . I n S inga p or e a lone , a r e c o r d num be r o f 2 179 c on f i r m e dcases of dengu e were repor ted in 1991.

Dengue v iruses compr ise four se ro logic types ( types 1 , 2 , 3 , and 4) andbe long to the f a m i ly F l a v i v i r i d a e w h o s e o t h e r r e l a t ed m e m b e r s i n c lu d e J a p a n e s ee nc e pha l i t i s, Kun j in , M ur r a y V a l le y e nc e pha l i t i s , St . Lou is e nc e pha l i t i s , t i c k -bo r ne e nc e pha l i t i s , W e s t Ni le , ye l low fe ve r, a nd he pa t i t i s C v i r u s e s. The d e nguev i ru s c o n s i st s o f a s i n g le p o s i t i v e -s t r a n d e d R N A g e n o m e o f a p p r o x i m a t e l y 11 k be nc od ing the c a ps id , m e m b r a ne a nd e nve lope p r o te in s , a s we ll a s non - s t r uc tu r a lp r o t e i n s i n c lu d i n g t h re e m a j o r p r o t e i n s k n o w n a s N S 1 , N S 3 , a n d N S 5 w h o s ef unc t ions a r e n o t y e t fu l ly e luc ida te d . The NS 3 ge ne i s one o f the m os t c ons e r ve dge ne s a m ong the f ou r de ngue v i r u s s e r o type s , a nd inde e d e ve n a m ong o the rm e m be r s o f the f l a v iv ir u s f a m i ly [ 5 , 34 ]. A p u ta t ive s e r ine p r o te a s e d om a ins p a n s t h e N - t e r m i n a l r e g i o n o f t h e N S 3 p r o t e in [ 2, 6 ] . L y i n g C - t e r m i n a l t ot h e p r o te a s e i s a n u c l e o ti d e t r i p h o s p h a t a s e / R N A h e l ic a se d o m a i n c o n s i s ti n g o f7 c ons e r ve d s e gm e n ts num b e r e d I , I a , I I - V I th ou gh t to be the nuc le o t idet r ipho s pha te b ind ing s it e s [-5 , 18] . The s e two dom a ins a r e invo lve d in p r oc e s s ingo f the v i r a l po lyp r o te in wh ic h i s a c r i t i c a l e ve n t in v i r a l e xp r e s s ion a nd r e p l i -c a t io n . F u r t h e r m o r e , m o n o c l o n a l a n t i b o d i e s p r o d u c e d a g a i n s t N S 3 p ro t e i n sh a v e b e e n s h o w n t o c o n f e r p a r t i a l p r o t e c t io n i n l a b o r a t o r y m i c e a g a i n s t l e t h a lc ha l l e nge wi th de ngue v i r u s e s [ 42 ] .

C e l l c u l t u re a n d i m m u n o l o g i c a l a s s a y s s u c h a s n e u t r a l i z a ti o n a n d h a e m a g -g lu t ina t ion inh ib i t io n te s t s a re e s ta b l i s he d a nd w ide ly e m p loye d f o r the d ia gnos i sa nd typ in g o f de ngue v i r u se s . How e ve r , the s e t e s ts a r e r e la tive ly t im e - c on s um inga nd c ons ide r a b ly l e s s s e ns i t ive tha n ne we r m o le c u la r b io log ic m e thods s uc h a sn u c le i c a c id h y b r i d i z a t i o n [ 2 1] a n d 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 ].S inc e r a p id d ia gnos i s o f de ngue v i r u s in f e c t ion is c r i t i c a l in pa t i e n t m a na g e m e n ta n d d i se a se s u rv e il la n c e, w e a d o p t e d t h e t e c h n i q u e o f c D N A a m p l i f i c a t io n b yPC R f o r the s e ns i tive ide n t i f i c a t ion o f de ngue v i ru s e s . W e r e po r t he r e the u s eo f a s ing le pa i r o f NS 3 - spe c if ic c ons e ns us p r im e r s f o r P C R wh ic h c a n de te c ta l l f ou r de ngue v i r u s type s , a s we l l a s o the r r e la te d f l a v iv i r u s e s . I n a dd i t ion ,s e qu e n c e d a t a o f th e P C R p r o d u c t s f r o m t h e v a r i o u s v i r a l t e m p l a t e s a r e co m -p a r e d a n d a n a l y z e d .

M at er i a l s and m et hodsVirus strains

Reference strains of dengue type 1 (Haw aii), type 2 (N ew Guinea C), typ e 3 (H 87), andtype 4 (H 241), yellow fever (17 D vaccine), Japanese e ncephalitis (Nakaya ma), and Ku njin(MRM 6tC ) viruses tested in this study were propagated in C 6/36 Aedes albopictus cells[25]. D engue virus serotype was confirmed by imm unofluorescent staining of viral antigenby type-specific mon oclonal a ntibodies.I so la tion o f v ira l and cy top lasmic R N A

Den gue virions were purified by precipitation in 7% polyethylene glycol (PE G ) and 2.3%NaC t, and centrifugation through a 30% sucrose cushion. Dengue viral RNA s were isolatedby phenol/chloroform extraction and ethanol precipitation [39].


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PCR and sequencing of NS 3 of dengue and other flaviviruses 159Cytoplasmic RNAs were isolated from virus-infected and non-infected cells by a rapidmethod described by Gough [19]. Briefly, cells were harvested, pelleted and resuspendedin 10 mM Tris-HC1 (pH 7.5), 0.15 M NaC1, 1.5 mM MgC12 and 0.65% Nonidet P-40. Aftercentrifugation, the cytoplasmic lysate was transferred to an equal volume of 7 M urea, 1%

SDS, 0.35 M NaC1, 10 mM EDTA, 10mM Tris-HC1 (pH 7.5). The mixture was extractedwith phenol/chloroform and precipitated with ethanol.The RNAs were quantitated with a UV spectrophotometer at 260 nm, where an opticaldensity of 1 corresponds to approximately 40 gg/ml.Amplification primers

Two 17-base oligonucleotide consensus amplimers, DV 1 (upstream) and DV3 (down-stream) as shown in Table 1, were designed based on highly conserved motifs within theNS 3 gene from the published nucleotide sequences of dengue virus types 2 [13, 20, 26], 3[36], and 4 [31]. DV 1 was designed on the basis of the conserved Gly-Thr-Ser-Gly-Ser-

T a b l e 1 . Nucleot ide and deduced amino acid sequences of consensus PCR primers:homology with published dengue and other flaviviral strainsP r i m e r / N u c l e o t i d e s e q u e n c e A m i n o a c i d s e q u e n c e R e f e r e n c es t r a i n ( a n d p o s i t i o n ) ( a n d l o c a t i o n )

D V I ( +) p r i m e r 5 ' G G R A C K T C A G G W T C T C C 3 "D I S 2 7 5 ( 4 8 9 9) * * C * * A * * T * * * * * * * * ( 4 9 1 5D 2 N G C ( 4 9 18 ) * * * * * C * * * * * * * * G * * ( 4 9 3 4D 2 J A M ( 4 9 1 8) * * * * * * * * * * * * * * * * * ( 4 9 3 4D 2 P R I 5 9 ( 4 9 18 ) * * * * * * * * * * * * * * * * * ( 4 9 3 4D 2 P D K ( 4 9 18 ) * * * * * * * * * * * * * * * * * ( 4 9 3 4D 3 H 8 7 ( 4 9 10 ) * * * * * * * * * * * * * * * * * ( 4 9 2 6D 4 8 1 4 6 6 9 ( 4 9 17 ) * * * * * * * * * * * * * * * * * ( 4 9 3 3J E S 9 8 2 ( 5 0 04 ) * * * * * A * * C * * C * * A * * ( 5 0 2 0K N M R M 6 1 C ( 4 9 87 ) * * * * * A * * * * * C * * G * * ( 5 0 0 3Y F I 7 D ( 4 9 76 ) * * C * * * * * * * * * * * * * * ( 4 9 9 2M V E ( 5 0 03 ) * * * * * * * * * * * * * * C * * ( 5 0 1 9T B E W E S T ( 4 9 85 ) * * * * * A * * * * * C A G C * * ( 5 0 0 1W N V ( 4 9 51 ) * * * * * * * * * * * * * * C * * ( 4 9 67 )H C V l ( 3 4 87 ) * * C T * C * * G * * G G G * * * ( 3 5 03 )H C V B K ( 3 8 19 ) * * C T * * * * G * * * G G * * * ( 3 8 35 )

G X S G X( 1 6 0 7 ) * * * * * ( 1 6 1 1 ) [ 1 8 ]( 1 6 0 8 ) * * * * * ( 1 6 1 2 ) [ 2 7 ]( 1 6 0 8 ) * * * * * ( 1 6 1 2 ) [ 1 4 ]( 1 6 0 8 ) * * * * * ( 1 6 1 2 ) [ 2 1 ]( 1 6 0 8 ) * * * * * ( 1 6 1 2 ) [ 3 ]( 1 6 0 6 ) * * * * * ( 1 6 1 0 ) [ 3 7 ]( 1 6 0 6 ) * * * * * ( 1 6 1 0 ) [ 3 2 ]( 1 6 3 7 ) * * * * * ( 1 6 4 1 ) [ 4 2]( 1 6 3 8 ) * * * * * ( 1 6 4 2 ) [ 1 2 ]( 1 6 2 0 ) * * * * * ( 1 6 2 4 ) [ 3 8]( 1 6 3 6 ) * * * * * ( 1 6 4 0 ) [ 1 3 ]( 1 6 2 5 ) * * * * * ( 1 6 2 9 ) [ 3 3 ]( 1 6 3 4 ) * * * * * ( 1 6 3 8 ) [ 4 ]( 1 1 6 3 ) * * * * * ( 1 1 6 7 ) [ 9]( 1 1 6 3 ) * * * * * ( 1 1 6 7 ) [ 4 3 ]

D V 3 ( -) p r i m e r 5 ' A A R T G I G C Y T C R T C C A T 3 'D V 3 ( +) s e q . 5 ' A T G G A Y G A R G C I C A Y T T 3 " M D E A HD I S 2 7 5 ( 5 3 52 ) * * * * * * * * * * * * * * * * * ( 5 36 8 ) ( 1 75 8 ) * * * * * ( 1 7 62 )D 2 N G C ( 5 3 68 ) * * * * * * * * * * * * * * * * * ( 5 3 84 ) ( 1 7 58 ) * * * * * ( 1 7 62 )D 2 J A M ( 5 3 68 ) * * * * * * * * * * * * * * * * * ( 5 3 84 ) ( 1 7 58 ) * * * * * ( 1 7 62 )D 2 P R I 5 9 ( 5 3 68 ) * * * * * * * * * * * * * * * * * ( 5 3 8 4 ) ( 1 7 58 ) * * * * * ( 1 7 62 )D 2 P D K ( 5 3 68 ) * * * * * * * * * * * * * * * * * ( 5 3 84 ) ( 1 7 58 ) * * * * * ( 1 7 62 )D 3 H 8 7 ( 5 3 63 ) * * * * * * * * * * * * * * * * * ( 5 3 79 ) ( 1 7 57 ) * * * * * ( 1 7 6 1)D 4 8 1 4 6 6 9 ( 5 3 67 ) * * * * * * * * * * * * * * * * * ( 5 3 83 ) ( 1 7 56 ) * * * * * ( 1 7 6 0)J E S 9 8 2 ( 5 4 5 8) * * * * * * * * * * * * * * * * * ( 5 4 74 ) ( 1 7 8 8) * * * * * ( 1 7 9 2)K N M R M 6 1 C ( 5 4 40 ) * * * * * * * * * * * * * * * * * ( 5 4 5 6) ( 1 7 89 ) * * * * * ( 1 7 9 3)Y F I 7 D ( 5 4 3 2) * * * * * * * * * * * * * * * * * (5 4 4 8) ( 1 7 7 2) * * * * * ( 1 7 76 )M V E ( 5 4 56 ) * * * * * * * * * * * * * * * * * ( 5 4 72 ) (1 7 8 7) * * * * * ( 1 7 9 1)T B E W E S T ( 5 4 44 ) * * * * * * * * * * * * * * * * G ( 5 4 6 0) ( 1 7 7 8) * * * * * ( 1 7 82 )W N V ( 5 4 04 ) * * * * * * * * * * * * * * * * * ( 5 4 20 ) (1 7 8 5) * * * * * ( 1 7 8 9)H C V l ( 3 9 43 ) T G T * * * * * * T G * * * * * C ( 3 9 59 ) ( 1 3 1 5) C * * C * ( 1 3 19 )H C V B K ( 4 2 75 ) T G T * * * * * * T G * * * * * C ( 4 2 91 ) ( 1 31 5 ) C * * C * ( 1 3 19 )

[18][ 2 7 ][14][21][3][ 3 7 ][32][42][12][38][30][33][4][9][43]

(+) Sense orientation; ( - ) anti-sense orientationI Inosine; K G/T; R A/G; W A/T; Y C/TX Any amino acidAsterisk indicates match


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1 60 V . T . K . C h o w e t al.P r o se que nc e ( a m ino a c ids 1608- 1613 o f de ngue v i r us t ype 2 ) whic h l ie s in t he se r inepr o t e a se dom a in [ 2 , 5 , 6 ] . DV 3 wa s syn the s iz e d ba se d on t he M e t - Asp- G lu- Ala - H i s - Phem o t i f ( a m ino a c ids 1758- 1763) c or r e spond ing t o c on se r ve d se gm e nt I I o f t he nuc l e o t i det r i pho sph a t a se /R NA he l ic a se dom a in I -5 , 18]. T he DV 1 /DV 3 p r im e r pa i r f l a nks a 470 bptarge t region.

cDNA synthesisS y n t he s is o f v i ra l c D N A t e m p l a te s w a s p e r f o rm e d u s i n g a G e n e A m p R N A P C R k i t (P e r ki n -E lm e r Ce tus ) i n a 10 g l r e ve r se t r a nsc r ip t ion r e a c t i on m ix tu r e c ons i s t ing o f 1 ng de ng uev i ra l RN A or 1 gg c y top l a sm ic R N A f r om v i r us - in f e c t e d c el ls ; 1 x buf f e r c on t a in ing 10 m MT r i s- HC1 ( pH 8 .3 ), 5 0r a m KC1, 5m M M gC12; 0 .5 o r 1 m M e a c h o f t he f our de ox yr ibo-nuc l e os ide tr i phospha t e s ; 1 U / g l R Na se i nh ib i to r ; 0 .5 o r 0.75 gM DV 3 down s t r e a m pr im e ror 2 .5 gM r a ndo m he xa m e r s o r 2 .5 Ia M o ligo( dT ) p r im e r s a n d 2 .5 U /g l M olon e y r ou t inel e uka e m ia v i r us re ve r se t r a nsc rip t a se ; i nc uba t e d a t r oo m t e m pe r a tu r e f o r 10 r a in [ r a nd omhexam ers or ol igo(dT ) pr imers only ] , 42 C for 15 ra in , 99 C for 5 rain , and 4 C for 5 min.

Potymera se chain reactionT o the 10 g l syn the s iz e d c D N A vo lum e wa s t he n a dde d 40 ~tl o f a PC R m ix g iv ing f ina lc onc e n t r a t i ons o f 1 x PC R buf f e r c on t a in ing 10 m M T r i s- HC1 ( pH 8 .3 ), 50 m M KC1, 2 m MM g C 12 ; 0 .1 o r 0 .1 5 g M D V 1 u p s t r e a m p r i m e r [ o r b o t h D V 1 a n d D V 2 p r i m e rs f o r c D N A spr im e d w i th r a ndo m he xa m e r s o r o l i go( dT ) p r im e r s ] ; a n d 2 .5 U /10 0 g l Taq polym e r a se .T h i s m ix tu r e wa s sub j e c t e d t o a n i n it i al 95 C f o r 1 r a in , f o l l owe d b y 30 c yc l e s o f 95 Cden atura t ion for 0 .5 min, 50 C annea l ing for 1 min, 72 C extension fo r 1 ra in , w i th a 1 minr a m p be twe e n e a c h s t e p o f e ve r y cyc le , a nd a f i nal e x t e ns ion o f 72 C fo r 10m in . 20% ofe a c h P C R p r o d u c t w a s r e s o l v e d b y e l e c t r o p h o r e s i s o n a n e t h i d i u m b r o m i d e - s t a i n e d 2 %a ga r ose ge l . Ser ia l t e n - f o ld d i l u t ions o f pur i fi e d de n gue v i r a l R N A r a ng ing f r om 1 ng t o0 .01 f g i n 10 gg /m l o f c a r r ie r ye a s t tR N A we r e sub j e c t e d t o c D N A syn the s is a nd P C R a sde sc r ibe d t o a sc e r t a in de t e c t i on se ns it iv i ty . 1 pg o f pur i f ie d v i r a l R N A wa s e s t im a te d t o b ea ppr ox im a te ly 105 c op i e s , de rive d f r om the c a l c u l a t i on o f 1 m ole ( o r 11 000 x 33 0g) o fde ngu e v i r al RN A be ing e qu iva le n t t o 6 x 1023m ole c u l e s. S t r i nge n t p r e c a u t ions w e r e ta ke nt o a v o i d c ro s s c o n t am i n a t i o n o f sp e c im e n s fr o m " c a r r y o v e r " D N A [ 2 7 ].

Isolation and amplification of PC R products from agarose gelsT o ob t a in spe c if ic te m pla t e s f o r d i r e c t s e que nc ing , P C R pr odu c t s o f s iz e 470 bp we r e e xc ise df r om a ga r ose ge ls , DN A e lu t e d a nd r e a m pl i fi e d . Br i e fl y , t he c u t ba n ds w e r e le f t i n U l t r a f r e e -M C tu bes wi th 0 .45 ~tm f i lte r s (Mi l l ipore) a t - 80 C for 10ra in, th aw ed an d cent r i fug eda t 14 000 r pm f or 5m in . 100 ~tl o f T E buf f e r ( 10 m M T r is - HC1 pH 8 .0, 1 m M E D T A ) w e r eadd ed to the cent r i fuga te , th en incu bate d a t 37 C for 15 ra in and cent r i fuged a t 14 000 rpmf or 5 m in . T he D N A in th i s m ix tu r e wa s e x t r a c t e d t h r ic e w i th bu t a no l a nd tw ic e w i th e ther ,e tha no l p r ec ip i t a te d , w a she d a nd r e suspe n de d in T E . A ppr o pr i a t e d i l u t i ons o f t he e lu t e dD N A w e r e r e a m p li fi e d b y P C R ( w i t h o u t r am p i n g ) a n d e l e c tr o p h o r e s ed a s d e s c ri b e d a b o v e .

Purification and direct sequencing of PC R productsT he r e a m pl i f i e d p r odu c t s we r e e x t r a c te d w i th c h lo r o f or m a nd e the r, p r e c ip i t a t e d w i th PE G ,N a C1 a n d w a s h e d w i t h 8 0 % e t h a n o l. A b o u t 0 .5 t o 1 p m o l o f e ac h P C R p r o d u c t t e m p l a t ewa s se qu e nc e d a t l e a s t tw ic e a nd i n bo th d i r e c ti ons w i th 32p 5 ' end- l a be ll e d D V 1 a nd DV 3p r im e r s u s i n g a d s D N A c y c le d id e o x y s e q u e n c in g s y s t e m ( 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 )a c c or d ing t o t he m a nuf a c tu r e r ' s i ns t r uc t i ons w i th m odi f i c a t i ons . Taq p o l y m e r a s e w a s e m -plo yed to ca ta lyse 20 l inear amp l i f ica t ion cyc les each a t 95 C for 0 .5 min, 50 C for 1 min


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PC R and sequencing of NS 3 of dengu e and othe r flaviviruses 161and 72 C for 1 min w itho ut ramping. The sequencing reactions were electrophoresed in8% polyacrylamide gels which were dried and autoradiographed.

Computer analysisNucle otide and amino acid sequences were aligned and com pared using the DNA SIS (eighthversion) and PROSIS (fourth version) software programmes (Hitachi).Results

PC R amplification of N S3 fragments o f dengue and other flavivirusesUsing p r imers DV 1 and DV 3 , ta rge t f r agmen t s o f expec ted s ize 470 bp cou ldbe success fu l ly ampl i f i ed on cD N A templa te s syn thes ized f rom pur i f i ed RN Asof dengue v i rus types 1 , 2 , 3 , and 4 as wel l as f rom cytoplasmic RNAs of ce l lsin fec ted wi th each o f the fou r dengue , Japanese encepha li t i s, Kun j in and ye l lowfever v i ruse s us ing speci fi c dow ns t r eam p r imers (F ig . 1 A) o r r an do m pr imers

Fig. 1. Gel electrophoresis o f PCR products of 470 bp amplified from cDN As synthesizedusing A specific downstream, C random, or D oligo(dT) primers from cytoplasmic RNAsof C 6/36 cells infected with deng ue types 1 (1), 2 (2), 3 (3), 4 (4), Kun jin (5), Japaneseencephalitis (6), yellow fever (7) viruses; and of n on-infecte d cells (8). M 123 bp marker.B Ream plification of excised 470 bp band s fr om A yields sharp target ban ds suitable fordirect sequencing


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162 v . T . K . C h o w e t a l.( F i g . 1 C ) . I n c o n t r a s t , u s i n g c D N A t e m p l a t e s d e r i v e d f r o m o l i g o ( d T ) p r i m e r s ,P C R y i e ld e d c l e a r ly d i s c e rn i b le b a n d s f o r a l l t h e v i r u se s t e s te d e x c e p t J a p a n e s ee n c e p h a l i ti s a n d K u n j i n v i ru s e s ( F i g . 1 D ) .

T h e p r e s e n c e o f e x t r a n e o u s b a n d s i n P C R p r o d u c t s u s in g s p e ci fi c d o w n -s t r e a m p r i m e r s f o r v i ra l c D N A s y n t h e s is ( F i g . 1 A ) m a y b e a t t r i b u t e d t o n o n -s p e ci fi c p r i m i n g o f t h e d o w n s t r e a m p r i m e r d u r i n g r e v e r se tr a n s c r i p t i o n a t al o w e r te m p e r a t u r e o f 42 C c o m p a r e d w i t h 5 0 C f o r P C R a n n e a l in g . H o w e v e r ,r e a m p l i fi c a t i o n o f e x c is e d 4 7 0 b p b a n d s ( in F i g . 1 A ) p r o d u c e d s h a r p t a r g e tf r a g m e n t s ( F i g . 1 B ) w h i c h s u b s e q u e n t l y y i e ld e d a u t h e n t i c v i r a l s eq u e n c e d a t a .

P C R p r o d u c ts g e n e ra t ed f r o m r a n d o m - p r im e d a n d o l i g o (d T ) -p r im e dc D N A s a s w ell a s t ho s e o b t a in e d f r o m P C R o f v ir al c D N A s s p i ke d w i th h u m a ng e n o m i c D N A y i e l d e d t a r g e t b a n d s w i t h o u t s i g n i f i c a n t n o n - s p e c i f i c b a n d s( Fig . 1 C a n d D ) . P C R o f h u m a n g e n o m i c D N A o r o f c D N A f r o m n o n -i n fe c te dC 6 /3 6 c e lls p r o d u c e d n o t a r g e t n o r b a c k g r o u n d b a n d s ( F ig . 1 A , C , a n d D , l a n e8 ) .

Sens i t iv i t y a s s ay s i nd i ca t ed t ha t a t l ea s t 1 , 0 . 1 , 0 . 01 , and 1 pg e qu i va l en t t o1 0 5 , 1 0 4 , t 0 3 , a n d 1 05 c o p i e s o f d e n g u e t y p e s 1 , 2 , 3 , a n d 4 v i ra l R N A s , r e -s p e c ti v e ly , c o u l d b e d e t e c t e d b y g e l e l e c t ro p h o r e s i s o f P C R p r o d u c t s ( F i g . 2 ).

Sequence analysis of N S 3 o f dengue and other flavivirusesC l e a r l y r e a d a b l e s t r e tc h e s o f ~ 4 2 0 n u c le o t i d e s ( e n c o d i n g 1 4 0 a m i n o a c i d re s -i du e s ) w it h in t h e N S 3 ta r g e t f r a g m e n t o f d e n g u e 2 ( N e w G u i n e a C ) , K u n j i n( M R M 6 1 C ) a n d y e l lo w f e v e r (1 7 D v a c c i n e ) v i ru s s t r a in s w e r e i d e n t ic a l w i t ht h e c o r r e s p o n d i n g p u b l i s h e d s e q u e n c e s [ 1 1 , 2 6 , 3 7 ] .H o w e v e r , 2 n u c l e o t i d e d i f f e r e n c e s w e r e n o t e d w i t h i n t h i s f r a g m e n t b e t w e e no u r s e q u e n c e f o r d e n g u e 3 ( H 87 st r a i n ) a n d t h e p r e v i o u s l y p u b l i s h e d s e q u e n c ef o r th e s a m e s t ra i n [ 3 6 ] , c u l m i n a t i n g i n a s in g le c o n s e r v a t i v e h y d r o p h o b i c a m i n oac i d d i f f e r ence a t r e s i due 1709 (F i gs . 3 an d 4 ) .

T w e n t y - o n e n u c l e o t i d e d i f fe r e n c e s w e r e o b s e r v e d b e t w e e n t h e N S 3 f r a g -m e n t s o f th e H a w a i i st r a i n a n d t h e p u b l i s h e d S i n g a p o r e S 2 7 5 / 9 0 s t ra i n [ 1 7 ]o f dengu e 1 v i r u s ( F i g . 3 ). The s e l ead t o 4 d i s pa r i ti e s a t am i no ac i d r e s i dues1688, 1690 , 1691 , an d 1729, l oca t ed be t w een t h e cons e r v ed s egm en t s I , I a an dI I w h i c h a r e n u c t e o t i d e b i n d i n g s i t e s w i t h i n t h e p u t a t i v e n u c l e o t i d e t r i p h o s -p h a t a s e a n d R N A h e li ca s e d o m a i n ( F ig . 4 ).D i f f e r e n c e s a t 1 6 n u c l e o t i d e p o s i t i o n s w e r e s ee n b e t w e e n t h e H 2 4 1 s t r a ina n d t h e p u b l i s h e d 8 1 46 69 s t r a in [ 3 1 ] o f d e n g u e 4 v i r u s, w i t h o n l y o n e r e s u l t a n tp o l a r v e r s u s h y d r o p h o b i c a m i n o a c i d r e p l a c e m e n t a t r e s i d u e 1 6 4 4 w i t h i n t h es e r i n e p r o t e a s e d o m a i n .T h e N a k a y a m a s t ra i n a n d t h e p u b li s h e d J a O A r S 9 8 2 s t ra i n [ 4 0 ] o f J a p a n e s eencep ha l i t i s v ir u s d i f f e r ed a t 11 nuc l eo t i de p os i t i ons , r e s u l t i ng i n on l y one am i noa c i d d i f f e r e n c e e m e r g i n g w i t h i n t h e s e r i n e p r o t e a s e d o m a i n a t r e s i d u e 1 6 6 2 .C o m p a r i s o n o f t h e N a k a y a m a s tr a in w i t h t he a t t e n u a t e d v a c c in e S A A s t r ai n[ 1 ] r e v e a l e d a n a d d i t i o n a l d i s p a r i t y a t a m i n o a c i d re s i d u e 1 7 3 9 l o c a t e d b e t w e e ns e g m e n t s I a a n d I I o f th e R N A h e li c as e d o m a i n ( F ig s . 3 a n d 4 ).


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PCR and sequencing of NS 3 of dengu e and other flaviviruses 163

Fig. 2. PCR produ cts amplified from ten- fold serial dilutions of purified viral RN As ofdengue virus A type 1, B type 2, C type 3, D type 4. cDNA templates for PCR weresynthesized from starting RN As of 1 ng, 0.1 ng, 0.01 ng, 1 pg, 0.1 pg, 0.01 pg, 1 fg, 0.1 fg,0.01 fg (1-9) using rando m primers. M 123 bp m arke r

DiscussionUs ing a s ingle pa i r of NS 3 consens us am pl imers , we are able to d e tec t al l fourdengu e v i rus types , a s we l l as Jap anese encepha li t i s, Kun j in and ye l low feve rv i ruse s by reve rse t r ansc r ip t ion o f v i ra l RN A and /o r cy top la sm ic RN A of v irus -in fec ted cel ls , fo l lowed by PC R, ach ievab le w i th in hours , cD NA s syn thes izedf rom ran do m and speci fi c dow ns t re am p r imers se rved a s re l i ab le t empla te s fo rP C R . A m p l i f i c a t io n o f o l i g o ( d T ) - p ri m e d c D N A s y i e ld e d t a r ge t p r o d u c t s f o ra l l the v i ruses tes ted except for Japanese encephal i t i s and Kunj in v i ruses . Suc-ce ss ful PC R f rom o l igo (dT) -p r im ed cD NA s f rom v i ra l t empla te s imp l i es thep re sence o f po lyadeny la ted s t ret ches w i th in the i r genomes . The h igh de tec t ionsens i t iv i ty o f the PCR p ro toco l u s ing the se consensus p r imers i s ev iden t , a sl i t tle as 0 .01 pg o r 103 copies of v i ral R N A being de tec ta ble on an agaros e ge la lone .

Even th oug h the se NS 3 ampl imers we re no t t e s t ed aga ins t o the r r e l a t edf l av iv iruses , comp ute r sea rch fo r p r im er h om olo gy wi th hepa t i t i s C I -9 , 41 ] ,


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164 V . T . K . C h o w e t a l.i 0 2 0 3 0 4 O 5 0

D I H A W G T G A A C A G A G A G G G A A A A A T A G T A G G T C T T T A T G G A A A T G G A G T G G T G A CD I S 2 7 5 ( 4 9 20 ) * * * * * * * * * * * A * * * * * e * * * * * e * * * e * * * * * * * * e * * * * * * * A * * * * eD 3 H 8 7 V A ' A * * * * * * * * * * * * * e G G * * * * G * * A * * G * * * * * C , e * * * * * * * * * T e *D 3 H 8 7 ( 4 93 1 ) A * A * * * * * * * * * * * * e e G G * * * * G * * A * * G e * * e * C * * * * * * * * * * * T * *D 4 H 2 4 1 . . .. . . * * G A * A * * * * * * G * T A * C * * A * * C * * C * * * * * * * * * * * A * * C * *D 4 8 1 4 6 6 9 ( 4 94 4 ) . . . .. . * * G A * A * * * * * e G * C A * C * * A * e C * * * * * * * * * * * * * * A * * T * *J E N A K . . . G * T T C T A * T * * e G * C * * C A * * * * C * * A * * C * * C * * * * * * * * T * A * C TJ E S 9 8 2 ( 5 0 28 ) . . . G * T T C T A * T * * * G * C * * C A * * * * C * * A * * C * * C e * * e * * * e T * A * C T

6 0 7 0 8 0 9 0 i 0 0D I H A W A A C A A G T G G A A C C T A C G T C A G T G C C A T A G C T C A A G C T A A A G C A T C A C A A GD I S 2 7 5 * * * * * * * * e * * * * * * * * * * * * * * * * * * * e * C * * * * * C * * * * * * * * * * * * *D 3 H 8 7 V * * A G * A * * * e G G * * * T * * T * * * * G A * * * * * G * * * A * A * * T * * * G A * * C * *D 3 H 8 7 * * A G * A * * * * G G * * * T * * T * * * * G A * * * * * G * * * A * A * * T * e * G A * * C * *D 4 H 2 4 1 T * A * T C A * * T G A T * * * * * * * * * * * T * * * A * G * * * * * C G * * A G * A * T G G T *D 4 8 1 4 6 6 9 C * A * T C A * * T G A T * * * * * * * * * * * * * * * A * G * * * * * C G * * A G * A T T G G * *J E N A K T G G C G * e * * C T ' A * * * * * * * * C * * * * * C * T G * * G * G * G * C C G T C A G G * G *J E S 9 8 2 T G G C G A * * * C T ' A * * * * * * * * C * * * * * C * T G * * G * G * G * C C G T C A G G * G *

i i 0 1 2 0 1 3 0 1 4 0 1 5 0D I H A W A A G G G C C T C T A C C A G A G A T T G A G G A C G A G G T G T T T A G G A A A A G A A A C T T AD I S 2 7 5 * * * * * e * c e * * * * * * * * * * * * * * * * * * * * e * * * * * * * * * * * * * * * * * * * *D 3 H 8 7 V * T * * A * * G A C * * * * * * * T e G * * A * * A * * * A * * * * C * A A * * G C * * * * T C * *D 3 H 8 7 * T * * A * e G A C * * * * * * * T * G * * A e * A * * * A * * * * C e A A * * G C * * * * T C * *D 4 H 2 4 1 * G C C A G A * T A T G A * * T * G A * * * * * * * - - - A * T * * * C * A * * G * A * * G A * * *D 4 8 1 4 6 6 9 * G C C A G A * T A T G A * * T * G A * * * e * * * - - - A * T * * * C * A * * G * A * * G A * * *J E N A K * G C C A G T C * C * G A * * C T T A C A C C C C A A * C A * * * * G * * A * * G * * * C * G A * GJ E S 9 8 2 * * C C A G T C * C * G A * * C T T A C A C C C C A A * C A * * * * G * * A * * G * * * C * G A * G

1 6 0 1 7 0 1 8 0 1 9 0 2 0 0D I H A W A C A A T A A T G G A C C T A C A T C C A G G A T C G G G G A A A A C A A G A A G A T A T C T T C CD I S 2 7 5 * * * * * * e * * * * * * * * * e * * * * * * * * * * e e , * * * * * * * * e * * * * * * * * * * eD 3 H 8 7 V * * C * * e * e * e * T * * T * * * * * T * * G * * A * * A * * G * * G C * G * A * * * * * * * * *D 3 H 8 7 * * C * * * * * * * * T * * T * * * * * T * * G * * A * * A * * G * * G C * G * A * * * * * * * * *D 4 H 2 4 1 * * T * * * * * * * * * T * * * * C * * C * * * G * C * * A * * G * * * * A * * * * A T * * * C * *D 4 8 1 4 6 6 9 * * T * * * * * * * * * T * * * * C * * C * * * G * T * * A * * G * * * * A * * * * A T * * * * e *J E N A K * * T G * G C * A * * T T * G * e C * * * * * T * * A * * * * * * * * C * * G * A * A T * * * G * *J E S 9 8 2 * * T G * G C * A * * T T * G * * C * * T * * C * * A * * * * * G * * C * * G * A * A T * * * G * *

2 1 0 2 2 0 2 3 0 2 4 0 2 5 0D I H A W A G C C A T A G T C C G T G A G G C T A T A A A A A G G A A G C T G C G T A C G C T A A T C T T G GD I S 2 7 5 * * * * * * * * * * * * * * * * * * C * * * * G * * * * * * C G * * * * C * * A * * * * * T * * * *D 3 H 8 7 V * * * T * * T * * T A * A * * * * * A * * C * * G * * A C G C T * A A * G * * T * * * * * T * * * *D 3 H 8 7 * * * T * e T * * T A * A * * * * * A * * C * * G * * A C G C T * A A * G * * T * * * * * T * * * eD 4 H 2 4 1 * T ' A * * * * * * A * A * * A * * C T * * * * * * * * * G * * * * * * A * * C T * G * * T * * * *D 4 8 1 4 6 6 9 * T * A * * * * * G A * A * * A * * C T * * * * * * * * * G * * * A * * A * e T T * G * * T * e A *J E N A K * C A A * * * A * T A A G * * C * * * e * T C * G C A * C G C * * A A * A * * A G C T G * G * * * *J E S 9 8 2 * C A G * * * A * * A A G * * C * * C * * C C * G C A * C G C * * A A * A * * A G C T G * G C * * *

D I H A WD I S 2 7 5D 3 H 8 7 VD 3 H 8 7D 4 H 2 4 1D 4 8 1 4 6 6 9J E N A KJ E S 9 8 2


2 6 0 2 7 0 2 8 0 2 9 0 3 0 0C T C C C A C A A G A G T T G T C G C T T C T G A A A T G G C A G A G G C G C T C A A G G G A A T G* * * * * * * * * * G * * * * * * * * * * * C * * * * * * * * * * * * * * * * * * * * * * * * * * ** A * * A * * * * * G * * A * * * * * A G * * * * G * * * * A * * * A * * A T * G * * A * * G C * C* A * * A * * * * * G * * A * * T * * A G * * * * G * * * * A * * * A * * A A * G * * A * * G C * C* * * * * * * G * * * * * G * * G * * G G * C * * G * * * * A * * * * * * C * * A C G T * * * C * ** * * * * * * G e * * * * G * * G e e G G * C * * G * * e e A * * * * * * C * e A C G T * * * C * ** A * * G * * G C * G * * G * * A * * A G ' A * * * * * * * * * * * A * * T T * G * G A * * G C e C* A * * G * * G C * G * * G * * A * * A G * A * * * * * e * * * * * A * * T T * G * G A * * G C * C

3 1 0 3 2 0 3 3 0 3 4 0 3 5 0C C A A T A A G G T A T C A G A C A A C A G C A G T G A A G A G T G A A C A C A C A G G A A G G G A* e e e e e w * * * * C * e A e e * e * * e e * e * * e * * * * * * e e e e e e * * e e e e A A e e* * * * * * * * * * * W * * A * * * * * T * * * A C A * * A T C * * * * * * * e * * * * * * * A * ** * * * * * * * * * * * * * A * * * * * T * * * A C A * * A T C * * * * * * * * * * * * * * * A * ** * * * * C C * T * * * * * * * * C C * * e * T * * * * * A T C A * * * * * * * * * * * * * * A * ** * * * * C C * T * * * * * * * * C C * * * * T * * * * * A T C A * * * * * * * * * * * * * * A * *e * e G * * C * A * * * * * A * * T T * * * * * * * * c e * * * A * * G * * * C A * . . . . . .. .* * * G * G C * A * * * * * A * * T T * * * * * * * * C * * * * A * * G * * * C A * . . . . . .. .


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PCR and sequencing of NS 3 of dengue and other flaviviruses 165D I H A WD I S 2 7 5D 3 H 8 7 VD 3 H 8 7D 4 H 2 4 1D 4 8 1 4 6 6 9J E N A KJ E S 9 8 2

3 6 0 3 7 0 3 8 0 3 9 0 4 0 0G A T A G T T G A C C T T A T G T G C C A T G C C A C T T T C A C C A T G C G T C T C C T G T C T C

* * * T * * * * * T * * A * * * * * T * * C * * A * * G * * * * * A * * * * * C T * G * * * * * A ** * * T * * * * * T * * A * * * * * T * * C * * A * * G * * * * * A * * * * * C T * G * * * * * A ** * * C * * A * * * * * C * * * * * T * * * * * A * * C * * * * * A * C A A * A * * T T * A * * A T* * * T * * A * * * * * C * * * * * T * * * * * A * * C * * * * * A * C A A * A * * T T * * * * A T . . . . , , . . . . . . . . . . . . o . , o . . . . . . . . . . . , . . . o . o . . . . . o , . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , . . . . . . , , . . o , . .


4 1 0 4 2 0 4 3 0C C G T G A G A G T T C C C A A T T A C A A C A T G . . . .* * * * * * * * * * * * * * * * * * * * * * * * * * . . . .* A * * C * * G * * * * * A * * C * * * . . . . . . . . . .* A * * C * * G * * * * * A * * C * * * . . . . . . . . . .* A A C C * * G * * * * * A * * * * * * * * * C * C A T A .* A A C C * * G * * * * * A * * * * * * * * * C * T A T A . . . . . . . . . o . . . o , . . . , . . o . . . . . . . . . . , . o , . . . . . . . o . . . , . o

Fig. 3. Com parison of our nucleotide sequences within the N S 3 genes of dengue 1 Hawaii(D1HA W), d e n g u e 3 H 8 7 (D3H87V),deng ue 4 H 241 (D4H241)and Jap anese encephalitisNakayama (J ENAK)strains with the published sequences of correspondingly-related den-gu e 1 $275/90 (D1S275),dengue 3 H 87 (D3H87),deng ue 4 814669 (D4814669)and Japaneseencephalitis $982 (JES982)strains. Num bers w ithin parentheses correspond w ith publishedpositions. A sterisks denote b ase matching with dengue 1 H aw aii strain, while dashes areintroduced for alignment. (Clearly readable ladders were obtained for only 338 upstreambases of the Nakaya ma strain)

t i c k - b o r n e e n c e p h a l i t i s [ 3 2 ] , M u r r a y V a l l e y e n c e p h a l i t i s [ 1 2 , 2 9 ] , a n d W e s tN i l e t - 4 ] v i r u s e s i n d i c a t e d m a t c h i n g o f h i g h p e r c e n t a g e w i t h t h e l a t t e r t w os u g g e s t i n g p o t e n t i a l a m p l i f i c a t i o n ( Ta b l e 1 ) .O t h e r w o r k e r s h a v e r e p o rt e d P C R a m p l i f i c a ti o n o f d e n g u e v i r u s e s a n d o t h e rf l a v i v i r u s e s u s i n g t y p e - s p e c i f i c p r i m e r s [ 1 4 , 1 5 , 2 4 , 3 5 ] a n d c o n s e n s u s p r i m e r s[ 2 2 , 2 8 ] f o r r e g io n s o u t s i d e o f N S 3, o f t e n a c c o m p a n i e d b y s e q u e n c e - s p e c if i cp r o b e h y b r i d i z a t i o n . P C R o f t h e s e R N A v i r u s e s u s i n g o u r t e c h n i q u e a n d t h ea s s a y s o f o t h e r g r o u p s i s c o m p a r a b l e i n t e r m s o f s e n s i t i v i t y , s p e c i f i c i t y , a n ds i m p l i c i t y t o P C R a s s a y s f o r D N A v i r u s e s s u c h a s h u m a n p a p i l l o m a v i r u s e s[ 1 0 , 4 3 ] .

T h e c o m p l e t e c o n c u r r e n c e o f o u r N S 3 f ra g m e n t s e q u e n c e d a t a w i t h t h ep u b l i s h e d s e q u e n c e s f o r t h e c o r r e s p o n d i n g s t r a i n s o f d e n g u e 2 , K u n j i n , a n dy e l l o w f e v e r v i r u s e s c o n f i r m s t h e a u t h e n t i c i t y a n d r e p r o d u c i b i l i t y o f t h i s R N A -P C R c o n s e n s u s am p l im e r t e ch n i q u e c o u p le d w i t h D N A s e q u en c in g .

S t r i n g e n t a n a l y s e s b y m u l t i p l e s e q u e n c i n g i n b o t h d i r e c t i o n s o f a m p l i f i e dc D N A t e m p l a t e s d e r i v e d f r o m r a n d o m , s p e c i f i c a n d o l i g o ( d T ) p r i m e r s w e r ec o n s i s t e n t l y c o m p a t i b l e . Th i s t h u s v a l i d a t e s t h e f i d e l i t y o f Taq p o l y m e r a s e f o rt a r g e t fr a g m e n t s o f l e s s t h a n 5 0 0 b p , i t s p r e v i o u s l y - v e r if i e d l o w e r r o r r a t e b e i n gc o m p a r a b l e t o e n z y m e s w i t h p r o o f - r e a d i n g a b i l i t y s u c h a s V e n t D N A p o l y -m e r a s e [ 2 3 ] .

I n t e r e s ti n g l y , th e m a j o r i t y o f t h e b a s e s u b s t i t u t i o n s f o u n d b e t w e e n d i f f e r e n ts tr a in s o f t h e s a m e v i r u s w e r e tr a n s it i o n s w h i c h a r e k n o w n t o b e t h e c o m m o n e s tc la s s o f p o i n t m u t a t io n s , w h i c h o c c u r a t r e la t iv e ly h i g h f r eq u e n c y a m o n g R N A


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1 66 V . T . K . C h o w e ta 1.

D I H A WD I S 2 7 5 ( 1 6 1 4)D 2 N G C 1 6 1 5 )D 2 J A M 1 6 1 5 )D 2 P R I 5 9 1 6 1 5)D 2 P D K 1 6 1 5 )D 3 H 8 7 VD 3 H 8 7 1 6 1 3 )D 4 H 2 4 1D 4 8 1 4 6 6 9 1 6 1 5 )J E N A KJ E S 9 8 2 ( 1 6 4 5)J E S A A ( 1 6 4 5 )K N M R M 6 1 C ( 1 6 4 5)Y F I 7 D ( 1 6 2 7 )

D I H A WD I S 2 7 5D 2 N G CD 2 J A MD 2 P R I 5 9D 2 P D KD 3 H 8 7 VD 3 H 8 7D 4 H 2 4 1D 4 8 1 4 6 6 9J E N A KJ E S 9 8 2J E S A AK N M R M 6 1 CY F I 7 D

$i 0 2 0 3 0 4 0 5 0

V N R E G K I V G L Y G N G V V T T S G T Y V S A I A Q A K A S Q E G P L P E I E D E V F R K R N L* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *I D K K * * V * * * * * * * * * * R * * A * * * * * * * T E K * I * D N - * * * * * D I * * * * K ** D * K * * V * * * * * * * * * * R * * A * * * * * * * T E K * I * D N - * * * * * D I * * * K R ** D K K * * V * * * * * * * * * * R * * A * * * * * * * T E K * I * D N - * * * * * D I * * * * R *I D K K * * V * * * * * * * * * * R * * A * * * * * * * T E K * I * D N - * * * * * D I * * * * R *I * * * * * V * * * * * * * * * * K N * G * * * G * * * T N * E P D * * T * * L * E * M * K * * * *I * * * * * V * * * * * * * * * * K N * G * * * G * * * T N * E P D * * T * * L * E * M * K * * * * . * K * * V I * * * * * * * * * K * * D * * * * * T * * E R T G * P D Y E V D * * - I * * * K R * . * K * * V I * * * * * * * * * K * * D * * * * * T * * E R I G * P D Y E V D * * - I * * * K R *D S - N * D * I * * * * * * * E L G G * S * * * * * V * G D R Q E * P V P E A Y T P N M L * * * Q MD S - N * D * I * * * * * * * E L G D * S * * * * * V * G D R Q E * P V P E A Y T P N M L * * * Q MD S - N * D * I * * * * * * * E L G D * S * * * * * V * G D R Q E * P V P E A Y T P N M L * * * Q M* D K N * D V I * * * * * * * I M P N * S * I * * * V * G E R M D * P V P A G F * P * M L * * K Q I* * * N * E V I * * * * * * I L V G D N S F * * * * S * T E V K E * * K E E L Q * I P T M L * K G M

I l a

6 0 7 0 8 0 9 0 i 0 0- T I M D L H P G S G K T R R Y L P A I V R E A I K R K L R T L I L A P T R V V A S E M A E A L K G- * * * * * * * * * * * * * * * * * * * * * * * * R * N V * * * * * * * * * * * * * * * * * * * * *- * * * * * * * * A * * * K * * * * * * * * * * * * * G * * * * * * * * * * * * * A * * E * * * R *- * * * * * * * * A * * * M e * * * * * * * * * * * * G * * * * * * * * * * * * * A * * E * * * R *- * * * * * * * * A * * * K * * * * * * * * * * * * * G * * * * * * E K * * A * * A * * E * * * R *- * * * * * * * * A * * * K * * * * * * * * * * * * * G * * * * * * * * * * * * * A * * E * * * R *- * * * * * * * * * * * * * K * * * * * * * * * * * * R * * * * * * * * * * * * * A * * E * * * * *- * * * * * * * * * * * * * K * * * * * * * * * * * * R * * * * * * * * * * * * * A * * E * * M * *- * * * * * * * * A * * * K * I * * S * * * * * L * * R * * * * * * * * * * * * * A * * E * * * R *- * * * * * * * * A * * * K * I * * S * * * * * L * * R * * * * * * * * * * * * * A * * E * * * R *- * V L * * * * * * * * * * K I * * Q * I K D * * Q Q R * * * A V * * * * * * * * A * * * * * * R *- * V L * * * * * * * * * * K I * * Q * I K D * * Q Q R * * * A V * * * * * * * * A * * * * * * R *- * V L * * * * * * * * * * K I * * Q * I K D * * Q Q R * * * A V * * * * * * * * A * * * * V * R *- * V L * * * * * A * * * * * I * * Q * I K * * * N * R * * * A V * * * * * * * * A * * * * * * R *T * V L * F * * * A * * * * * F * * Q * L A * C A R * R * * * * V * * * * * * * L * * * K * * F H *

i i 0D I H A W M P I R Y Q T T A VD I S 2 7 5 * * * * * * * * * *D 2 N G C L * * * * * * P * ID 2 J A M L e * * * * * P * ID 2 P R I 5 9 L * * * * * * P * ID 2 P D K L * * * * * * P * ID 3 H 8 7 V L * * * * * * * * TD 3 H 8 7 L * * * * * * * * TD 4 H 2 4 1 L * * * * * * P * *D 4 8 1 4 6 6 9 L * * * * * * P * *J E N A K L * V * * * * S * *J E S 9 8 2 L * V * * * * S * *J E S A A L * V * * * * S * *K N M R M 6 1 C L * * * * * * S * *Y F I 7 D L D V K F H * Q * F

1 2 0 1 3 0 1 4 0 1 5 0K S E H T G R E I V D L M C H A T F T M R L L S P V R V P N Y N M . . . .. . .* * * * * * K * * * * * * * * * * * * * * * * * * * * * * * * * * .. . .. ..R A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * L I . . .. ..R A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * L I. . . .. .R A * * * * * * * * * * * * * * * * * * * * * * * I * * * * * * L I . . . . . .R A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * L I . . .. . .* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . . . . . . .. .

* * * * * * * * * * * * * * * * * * * T * * * * S T * * * * * * L I . . .. . .* * * * * * * * * * * * * * * * * * * T * * * * S T * * * * * * L I . . .. . .Q R * * Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Q R * * Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Q R * * Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A R * * N * N * * * * V * * * * * L * H * * M * * H * * * * * * L F . . .. . .S A H G S * * * V I * A * * * * * L * Y * M * E * T * * V * W E V I . . .. . .

Fig . 4 . Com pa r a t i ve a na lys is o f de duc e d a m ino a c id r e s idue s c or r e spond ing t o nuc l e o t i dese que nc e s i n F ig . 4 . Kno wn se que nc e s o f de ngue 2 N G C (D2NGC),J A M (D2JAM), P R 1 5 9(D2PR159), a n d P D K (D2PDK), Ja pa ne se e nc e pha l i t i s SAA (JESAA), K u n ji n M R M 6 1 C(KNMRM61C) a nd ye l l ow f e ve r 17D (YF17D) s t r a ins a r e a l so i nc lude d . Num be r s w i th inpa r e n the se s r e f e r t o pub l i she d l oc a t i ons . As t e r i sks r e pr e se n t r e s idue s i de n t ic a l t o d e ngue 1Ha w a i i s tr a in a nd da she s pe r m i t op t im a l a l ignm e nt . S Co nse r ve d se r ine p r o t e a se m o t i f; LIa R N A he l ic a se se gm e nt I a nd I a m ot i f s


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PC R and sequencing of NS 3 of dengue and other flaviviruses 167v i r u s e s , p a r t i a l l y a t t r i b u t a b l e t o r a n d o m p o l y m e r a s e - m e d i a t e d b a s e m i s i n c o r -p o r a t i o n s [ 3 0 ] .

W e f ound 2 nuc le o t ide d i f f e r e nc e s be twe e n ou r NS 3 s e que nc e a nd the pub -l i s he d one f o r de ngue 3 ( s t r a in H 87 ). Th i s d i s c r e pa nc y m a y be pa r t ly e x p la ine dby d i f f e re nc e s in pa s s a ge h i s to r y o f th i s v i r a l s t ra in . O ur H 87 s t r a in wa s pa s s a ge d114 times in su ckl ing mo use bra in fo l lowe d by 5 t imes in C 6 /36 ce lls.

W h i l s t c o m p a r a t i v e s e q ue n c e a n a l y s i s o f s t r a in s o f d e n g u e 1 , d e n g u e 4 , a n dJ a pa ne s e e nc e pha l i t i s v i ru s e s re ve a le d num e r ous nuc le o t ide d i f f e r e nce s (21, 16,a nd 11 r e s pe ct ive ly ) , the s e we r e no t c o m m e ns u r a te w i th the a m ino a c id r e siduedif fe rences (only 4 , 1 , an d 1 respec t ive ly) . Al l these d if fe rences occur outs id eo f t h e t i g h t l y c o n se r v e d p u t a t i v e a c ti v e m o t i f s o f t h e e n z y m a t i c d o m a i n s w i t h i nN S 3, a n d m a n y m u t a t i o n s a r e s i le n t o r r e s u l t p r e d o m i n a n t l y i n c o n s e r v a ti v ea m i n o a c id s u b s t i t u t io n s . M o r e o v e r , t h e a b i l i t y o f o u r c o n s e n s u s p r i m e r s t oa m p l i f y t h is N S 3 f r a g m e n t o f d e n g u e a n d o t h e r f l a v iv i ru s e s f u r t h e r d e m o n -s t ra t e s t h e h i g h l y c o n s e r v ed n a t u r e o f t he s e m o t if s . F u r t h e r m o r e , t h e a p p a r e n ta bs e nc e o f d i f fe r e nc e s a t c ons e r ve d m o t i f s o f the s e r ine p r o te a s e a nd R N Ahe l ic a s e dom a ins i s c ons i s t e n t ly obs e r ve d e ve n f o r ge og r a ph ic a l ly d i s t inc t a ndte m por a l ly un r e la te d s t r a in s ( F ig . 4 ). The s e phe no m e na r e i t e r a te the s low r a teo f e vo lu t ion o f N S 3 ge ne s in f l a v iv ir u s e s , e s pe c ia l ly the c a ta ly t i c s i t es , r e in f o r c ingthe n o t io n o f the i r ind i s pe ns ib i l i ty in f l a v iv i r a l p r oc e s s ing a nd r e p l i c a t ion . I nc on t r a s t , h ighe r m o le c u la r e vo lu t iona r y r a te s ha ve be e n e s t im a te d f o r the e n -v e l o p e / N S 1 g e n e j u n c t i o n s o f d e n g u e v i r u s ty p e s 1 a n d 2 [ 3 8 ] , t h e p r e - M g e n eo f J a pa ne s e e nc e pha l i t i s v ir u s [ 8 ] , the VP 1 a nd 2 A ge ne s o f po l iov i r u s typ e 1[ 3 9 ] , t h e N P a n d M g e n e s o f p a r a m y x o v i r u s e s [ 3 3 ] , a n d f o r t h e r e g u l a t o rys e q ue n c es o f h u m a n p a p i l l o m a v i r u s t y p e 1 6 D N A [-7 , 2 3 ].

O u r d a t a o f fe r s o m e i n s i g h t i n t o t h e s t r u c t u r e - f u n c t io n a n a l y s i s o f N S 3 a n da l so d e m o n s t r a t e t h e f e as i b il i ty o f th e s t r a t e g y o f c o u p l i n g t h i s R N A - P C Rc o n se n s u s a m p l im e r t e c h n i q u e w i t h di re c t D N A s e q ue n c in g o f P C R p r o d u c t sin the m o le c u la r d ia gnos i s , e p ide m io logy a nd ve c to r s u r ve i l l a nc e f o r in f e c t ionswi th de ng ue a n d o the r f l a v iv i r a l s t r a in s . I n o r de r to type de ngue v i r u se s , in te r n a ltype - s pe c if i c p r im e r s f o r a l l 4 de ngue v i r u s type s in a ne s te d PC R f o r m a t a r ec u r r e n t ly be ing inve s t iga te d .

AcknowledgementsThis study was supported by a National University of Singapore grant (RP9t0438).C.L.K.S. is an N US research scholar.

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170 V .T .K . Cho w e t a l. : PC R and sequenc ing o f NS 3 o f f lav iv iruses41 . T akam izawa A , Mor i C , Fuk e I, Man abe S , Murak am i S, Fu j i t a J , On i sh i E , And ohT, Yo sh ida I , Ok ayam a H (1991) S t ruc tu re and o rgan iza t ion o f t he hepa t it i s C v i rusgenom e iso l a ted f rom hu m an ca r ri ers. J V i ro l 65 :1105-111342 . T an C HC , Ya p E H, S ingh M, D eube l V , Char t YC (1990) Pass ive p ro t ec t ion s tud iesin mice wi th mon oc lona l an t ibod ies d i rect ed aga ins t t he non-s t ruc tu ra l p ro t e in N S 3of dengue 1 v irus . J Gen Vi ro l 71 :745-7494 3 . T h a m K M , C h o w V T K , S i n g h P , T o c k E P C , C h i n g K C , L i m - T a n S K , S n g I T Y ,Berna rd H U (1991) Diagnos t i c sensi ti v it y o f po lymerase cha in reac t ion and Sou th e rnb lo t hybr id i za t ion fo r t he de t ec t ion o f hum an pap i l l omav i rus DN A in b iopsy specimensfrom cervical les ions. Am J Cl in Pathol 95:638-646

Au thors ' add ress : Dr . V . T . K . Chow , Dep ar tm en t o f Mic rob io logy , Facu l ty o f Med-ic ine , Nat ional Universi ty of S ingapore , Kent Ridge, S ingapore 0511, Republ ic of S in-gapore .Received October 20, 1992

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