Using improved non ri maprec assay to detect virulence mutations in poliomyelitis vaccine viruses advantages over rcta 40 test

E-mail: tthanh@vnmilk.com.vn 145 activity... David Evans, and Jeffrey W .A lm ond.. Philip M inor, Poliovirus vaccination: current understanding o f poliovirus interactions in hum ans an

V N U Jo u m a l of Science, N a tu ra l Sciences a n d T echnology 23 (2007) 145-151

ưsing improved Non Ri -Maprec assay to detect virulence mutations in poliomyelitis Vaccine viruses: advantages over

Thieu Thi Hoai Anh*, Le Thi Luan

Poliomyelitis vaccine Research and Production center (Poliovac), 135 Lo Duc, Hanoi, Vietnam

R eceived 23 Jan u ary 2006

A b s tr a c ỉ M u tan t an a ly sis by p o ly m erase Chain re actio n a n d re stric tio n en z y m e cleavage

(M A P R E C ) h as been d ev e lo p c d b y C hum ak o v et aỉ for p o lio v iru s to d e te rm in e q u a n tita tiv e ly the

p resence o f g en o m ic ch a n g es in p artic u la r nucleotide seq u e n ces th a t lead to v ừ u le n c e ph en o ty p es

o f oral p o lio m y e litis v accin e (O P V o r live attenuated v a c c in e ) [1] A fte rw ard s, this RI

(rad io iso to p e ) M A P R E C w as d e v e lo p e d to n o n RI - M A P R E C b y Ja p a n P o lio m y elitis R esearch Institute in 1998 to co m p en sa te fo r som e disad v an tag es o f R I-M A P R E C [2] T h is te st w as highly

reco m m en d o d fo r lab o ra to ries to assess the sa íe ty o f their v a c c in e P ro d u cts A t P o lio v a c, th e rctA0

test w as u se d to be c o n sid ered a m ajo r to o l for vaccine p ro d u c ts ex a m in a tio n In th is research , we

h ave im p ro v ed the n o n RI M A P R E C test to app ly it w ell w ith p ra c tic a l c o n d itio n s in our

lab o rato ry an d c o m p ared this test to the usual rct40 test to p o in t o u t its g re a t ad v an tag es.

1 I n tr o d u c tio n

P o lio m y elitis is an acu te d isease o f the

C entral nerv o u s sy stem (C N S ) cau sed by

poliovirus O ral p o lio m y elitis v acc in e (O P V ) is

usually co n sid ere d the m a jo r tool for

eradication o f p o lio m y elitis in m an y countries,

as w ell as V ietnam It is reg ard ed a s one o f the

safest vaccines in cu rren t use, b u t som e polio

vaccine related - p araly tic cases have been

reported recen tly [3] T h ese cases are cau sed by

vaccine v iruses u n d erg o in g rev erted m utations

during rep eatcd rep licatio n s

P olioviru s h as a g en o m e o f sin g le-stran d ed

RNA, its rate o f m u tatio n s is u su ally high

during rep licatio n sin c e R N A -d e p en d en t R N A

po lym erase does n o t h av e the p r o o f - read in g

* Corĩesponđing author Tel.: 84-4-8531213

E-mail: tthanh@vnmilk.com.vn

145

activity T h u s stric t con tro l is stro ng ly requ ired during v acc in e p ro d u ctio n

A lth o u g h th e re are m a n y alteratio n s

b etw een v iru le n t an d n o rm al íorm s o f

p o lio v iru ses: for S ab in type 1, 2 and 3, vaccine

strains d iffe r fro m th e ir p ro g e n ito rs by 55, 23 and 11 m u ta tio n s, resp ectiv ely [4], few changes req u ired fo r a tte n u a tic n and rev ersio n T h at is the reason w h y ty p e 3 S abin strain has the least stable g en o m e an d thus c an easily rev ert to viru len t fo rm w h ile S abin ty p e 1, in contrast,

h as the m o st stab le gen om e It w as

dem onstrated that neurovirulence increased

w hen the fo llo w in g ch an g es to o k Dlace in base

p o sitio n s o f th e v iral g en om e: in serotype 1,

p o sitio n 4 8 0 in the non c o d in g reg io n (N C R )

ch an g ed fro m G to A, p o sitio n 525 chang ed íro m u to c a n d th e re ío re c o m p en sa ted for the presen ce o f G -4 8 0 b y re s to rin g b ase-p airin g in

146 T.T.H A nh, L.T Luan / V N U Ịoum aỉ o f Science, Natural Sciences and Technology 23 (2007) 145-151

the stem -loop stru cture [5]; fo r serotyp e 2,

positio n 481 ch ang ed from A to G , p ro h ib itin g

the íb rm atio n o f a n ew b ase p a ir b etw een

residu es 481 and 511 th a t w eaken s the

second ary structu re [5]; fo r ty p e 3, p o sitio n 472

chang ed from u to c, resto rin g the

p oly pyrim iđin e tract-b in d in g p ro tein (an

initiation factor in n eu ro b la sto m a cells [6]) site

A ll these n u cleo tid es lay w ith in th e reg io n

design ated “in tem al rib o so m a l en try site”

(IR E S ), o f w h ich th e stab ility has a

co nsiderable in ílu en ce on th e translation

efficiency o f v iru s-sp eciíĩc p ro tein s associated

w ith n eu rov irulen ce, in o th er w ord s, these

m u tatio ns interfere w ith the IR E S ab ility to

in teract w ith /ra/w -actin g factors

C hu m ako v et al. in tro d u ced a m ethod

design ated “M u tan t an aly sis b y p o ly m erase

Chain reaction and restriction enzym e cleavage”

(M A P R E C ) to estim ate the ratio o f viruses

contain in g genes o f a v iru len t n atu re in a

vaccine virus p o p ulation W h en the p ro p o rtio n s

o f such genes ex ce ed a p articu la r level, the

vaccine fails the m o n k ey n eu ro v iru len ce test

(M N V T ): for type 1, ap p ro x im ate ly 5% o f 480-

A and 525-C co m bined; fo r ty p e 2, betw een

1.7% and 3.7% o f 48 1-G ; fo r ty p e 3, abo ut 1%

o f 472-C A t presen t, M A P R E C can b e u sed as

a su pplem ent to the M N V T for several

purposes: estab lish in g and m o n ito rin g o f

m olecular con sisten cy o f vaccin e p ro d u ctio n , as

a prelim inary m o lecu la r te st b efo re M N V T , as a

substitute o f the rct40 test, screen in g o f single

harvests an d m onito ring th e genetic stability o f

vaccine viruses

T he no n-R I M A P R E C te st w as d ev elop ed

from R I-M A P R E C b y Jap an P o lio m y elitis

R esearch Institute in 1998 to co m p en sate for

the b igg est d isad van tage o f R I-M A P R E C : its

use o f radioisotope re q u ire s a h ig h level o f

ex pertise an d the use o f eq u ip m en t, w hich,

unlike in eco n o m ically d e v e lo p e d co un tries,

m ay n o t be available in d ev elo p in g co un tries

w h ich a re th rea ten ed th e m o st by po lio m y eli tis

A íu rth e r co n sid era tio n is th at its use carries the risk o f c a u s in g en v iro n m en ta l p ro b lem s [2]

T h e rct40 test (rep ro đ u ctiv e cap acity at

4 0 ’C ) is u sed to be co n sid ered a sim ple tool for assessin g th e v iru len ce o f vaccine viruses via their te m p eratu re sen sitiv ity T h e re v e rta n t is

n o t te m p e ra tu re - sensitiv e, th e re ío re its rep ro d u ctiv e cap a city h a s no differen ce at th e tem p eratu res o f 36°c and 40°c T hus, by

ex am in in g th e rep ro d u ctiv e cap acity o f v accin e

v iru ses at th e se tem p eratu res and co m p arin g the resu lts, th e g en etic m aterial o f th e v accin e can

b e in d irectly assessed H o w ev er, m u tatio n s th a t have the g re a te st c o n trib u tio n to viru lence h av e

o n ly m in o r effect on viral te m p eratu re sen sitiv ity b u t o th e r n u cleo tid e s (6203, 7 07 1 ,

7410 an d 7441 for typ e 1; 20 34 for type 3) [4,5,7] T h e re ío re , i f a single h arv est sh o w s tem p eratu re sen sitiv ity , it d o es not m ean to tally this sam p le is n o t neu ro v iru len t T h is g reat

d isad v an tag e o f rc /4 0 te st can be d e íin ite ly

o v erco m e b y M A P R E C

In th is stu d y , w e p e ríò rm e d im p ro ved n o n

R I-M A P R E C assay on ty p e 3 vaccine viruses,

w h ich h av e th e g reatest p o ssib ility o f rev ertan t

m utatio n to assess th e ir sa íe ty and co m p ared this test to the usu al rctAO test in term s o f effectiv en ess, ex actitu d e, tim e co nsum ed and

ex p en ses O n the b asis o f these resu lts, w e reco m m en d ed One o f th e se tw o tests for the in vitro e x a m in a tio n o f oral p o lio v a c c in e ’s safety

in o u r la b o rato ry

2 M a te r ia ls a n d m e th o d s

2.1 Viraỉ RNA extraction and cDNA synthesis

In th is stu d y w e u sed six type 3 p o lio virus single h arv ests p ro d u ced in 2005 at Poliovac

d esig n ated 40 2, 403, 40 4, 405, 406, 407 and 4

T.T.H A nh, L.T Luan / V N U lo u m a l o f Science, Natural Sciences and Technology 23 (2007) 145-151 147

m a rk e r sam p les d esig n ated 472 -a, 4 7 2 -b , 472-c,

4 7 2 -d p ro v id eđ b y Jap an P o lio m y elitis

R esearc h Institute

P ositive sin g le -stran d ed R N A o f p olio v iru s

w a s ex tracted fro m 0.5 m l v iru s su sp e n sio n by

p h enol ex tractio n w ith so dium d o d ecy l sulfate

(S D S ) o f final co n ce n tratio n o f 0 5 % T he viral

R N A w as p recip ita ted u sing 100% isop rop ano l

an d treated w ith ethanol 70% , cD N A w as then

sy n th esized w ith M o lo n ey m u rin e leukem ia

viru s reverse tran scrip tase (M M L V -R T -

In v iừ o g en ), ran d o m h ex an u c leo tid e p rim ers

(F erm en tas) an d in cu bated for l h at 37"C

2.2 MAPREC, non Rl-MAPREC and improved non RỊ-MAPREC

B o th M A P R E C an d non R I-M A P R E C are

qu ite id en tical ex cep t for the w ay th ese tw o

m eth od s u sin g to q u an tify d ig ested D N A bands

F o r p o lio v iru s ty p e 3, a v iru len t b ase c at resid u e 4 7 2 w as ex am in ed in our research

S in ce n o k n o w n restric tio n en zy m e cuts either atten u ated o r rev erted p o lio v iru s sequences at

n u cleo tid e 4 7 2 , a restric tio n site o f Mbol is

en g in eered b y P C R u sin g a m u tagen ic prim er

T h u s the re stric tio n site is co m p o sed , in part, o f the p rim e r seq u en ce and in p art, o f the viral

cD N A u se d as a tem plate

T a b le 1 P C R p rim ers u se d to detect m u tatio n s b y M A P R E C and non R I-M A P R E C

♦MAPREC

P rim er p o la rity P rim e r seq u e n c e

sense T 4 3 1G A G C T A C A T G A G A G T g C T C C G G C C C C T G A A T G C G G C T g A 4 7 0

an tisen se C 5 13 A G G C T G G C T G C T G G G T T G C A G C T G C C T G C 4 8 4

L o w ercase letters sh o w m o d iíĩc atio n s

c o m p ared to th e p o lio v iru s seq u en ce w hich

w ere introduced to create o r d e stro y restrictio n

sites T h e norm al v accin e seq u en ce w as cut by

Hinĩl, the rev ertan t seq u en ce w as c u t b y MboI

♦ n o n R I-M A P R E C

A g at p o sitio n 4 4 7 w as in tro du ced to destroy restrictio n site for Hinữ site w hile a g at

p o sitio n 4 6 9 w as in serted to create restriction sites for b o th Hi nữ and Mbol.

P rím er p o la rity P rim e r sequ en ce _

sense T 4 4 0 G A G A G T C C T C C G G C C C C T G A A T G C G G C T g A T 4 7 1

an tisen se A 5 3 2 C G G A C T T G C G C G T T A C G A C A G G C T G G C T G C 5 0 2

ư n lik e M A P R E C , in n on R I-M A P R E C ,

on ly Mboỉ w as u sed to cu t th e revertan t

sequence P C R am p liíĩcatio n u sin g sense and

an tisense p rim ers as d escrib e d p rev io u sly and

Taq D N A p o ly m e ra se (In v iừ o g e n , P erkin-

E lm er) w as c o n d u cted for 40 cycles

F o r R I-M A P R E C , P C R am p liíic a tio n w as

asym m etric (i.e a 1 0-fold e x c e ss o f sense

p rim er ensu res th a t the p re d o m in a n t p ro d u ct o f

this reactio n is sin g le -stran d ed D N A : 30|Jg/m l

for sense p rim er an d 3 n g /m l fo r a n tise rse

prim er) T h e seco n d stran d is syn thesized using

a 32 p la b eled an tisen se prim er A fter treatm ent

o f the a m p liíie d D N A p ro d u ct w ith Mboỉ, the

d ig ested m aterial w as separated by elec ừ o p h o re sis in p o ly a cry lam id e gel T he

m u tatio n al ch ang e (p ercen tag e o f 472C ) w as calcu lated by m e asu rin g rad io activ ity in counts

p er m in u te (cp m ) o f d ig ested an d undigested

b an ds, u sin g th e equ ation : d ig e sted D N A band (cpm ) o v e r d ig ested D N A b an d (cpm ) plus

u n d ig e sted D N A b an d (cpm )

148 T.T.H A nh, L.T Luan / V N U Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 145-151

F or n o n R I-M A P R E C , P C R am p liíĩcatio n

w as perfo rm ed u sing th e p rim ers show n in

T ab le 1, w ith sense an d an tisen se p rim ers at

equ im olarity (b oth at a íinal co n cen tratio n o f

3 ụ g /m l) to iíave the PC R p ro d u ct o f 93bp A fter

treatm en t o f 25^1 o f the D N A p ro d u ct w ith the

com position o f Mboỉ and its b u ffer

(F erm entas), the d ig ested m aterial co n sistin g o f

D N A ban d s o f 30bp and 63 bp w as ap p lied to a

12% po ly acry lam id e gel to g e th er w ith 6X

loading bu ffer, and e lectro p h o resed w ith IX

T A E b u ffer o r 0.5X T B E b u ffe r for 2h at room

tem perature, u sing 100V A fìerw ard s, the gel

w as stained for 30m in w ith S Y B R G reen 1

(S igm a) đilu ted 10000 - f o ld w ith T A E o r T B E

T h e dig esíed D N A b a n d s w ere d etecteđ by

irradiating w ith u v ray s at a w av elen g th o f

250 nm and th e ir q u an tities w ere d eterm in ed by

the use o f a d u al-w av elen g th ch ro m ato scan n er

T o g eth er w ith 4 groups o f reco m b in an t m arker

viruses d esig n ated 47 2-a, 472-b, 472-c, 472-d,

for w hich th e p ercen tag e o f 4 7 2 -C values

(determ ined by R I-M A P R E C ) w ere 0 72% , 1%,

2.8% and 4 0% , resp ectiv ely , the calibration

curve w as prep ared , in w h ich the axis o f

ab scissa in dicates the A uorescence in tensity o f

digested D N A b an d s and the axis o f ordinate

indicates the p ercen tag e q u an tities o f 472-C

ư s in g this calib ration curve, w e can determ in e

th e 472-C p ercen tag e o f eac h sin gle h arv est

based on th e A uorescence in te n sity o f dig ested

D N A b a n d s and d ecid e w h e th e r it passes the

M N V T o r not

F o r im p ro v ed n o n R I-M A P R E C ap p lied in

ou r lab o ra to ry , w e co m p ared the electro p h o resis

p a tte m o f v irus sam p les to th e m a rk e r virus groups A sam p le p a sse s the te st i f no 63bp and 30bp d ig e sted D N A b an d s w o u ld ap p ea r or they

ap p ear b u t the b a n d s are w e a k e r than the

c o ư e s p o n d in g b an d s o f 4 7 2 -b m a rk e r (472C

p erce n tag e o f 1%) - like the p a tte m o f 472-a

m ark er (4 7 2 C p erce n tag e o f 0 7 2 % ) F or any case th at is h ard to co m p are b y u n aid ed eyes,

th e sam p les sh o u ld be re te ste d b y rcí40 test

3 R e s u lts

Fig 1 sh o w s the p a tte m obtain ed by

e le c tro p h o re sis in an aly zin g ty p e 3 polio viruses and 4 g ro u p s o f re c o m b in a n t m a rk e r viruses

A ll the v a c c in e sam p les p ro d u c e d at Poliovac in

2005 sh o w n o 30 bp an d 6 3 b p b a n đ s w hile the

c o rre sp o n d in g b an d s o f 4 7 2-b , 4 7 2 -c , 472-d are obv io us T h e 6 3 b p b a n d o f 4 7 2 -a is hard to

d etect sin c e its p ro p o rtio n o f 4 7 2 C is ju st 0.72% AU th e ty p e 3 sin gle h a rv e sts pass the non R I-M A P R E C and are safe fo r use

F ig 1 E lectro p h o resis p a tte m o f type 3 p o lio v iru s b y n o n R I-M A P R E C w ith p B R 3 2 2 /A /jp I la d d e r, arrows indicate 90bp, 67bp and 30 b p b an d s 1 , 2 , 3 , 4 , 5, 6, 7 are v im s sa m p le s (sin g le h a rv e sts) d e sig n a te d 402, 403,

4 0 4 ,4 0 5 ,4 0 6 , 4 0 7 , re sp ectiv ely 8, 9, 10, 11 are 472-d, 4 7 2 -c , 4 7 2 -b an d 4 7 2 -a , re sp ectiv ely

T.T.H A nh, L Ĩ Luan Ị V N U Ịournal o f Science, Natural Sciences and Technology 23 (2007) 145-Ĩ5Ĩ 149

It th e re ío re can be affirm ed th at M A P R E C

test is b o th e ffe c tiv e and exact W e also

co m p ared th is te st to the usual rctAỒ test in

term s o f tim e c o n su m e d and ex pen ses

T able2 C o m p a riso n b etw e en the tw o tests in term s

o f tim e c o n su m e d an d ex p e n ses (p e r 1 sam ple)

T im e c o n su m e d E xpenses

n o n R I-M A P R E C te st 25h -1 0 Ư S D

So, co m p a re d in term s o f tim e con sum ed

and expen ses, im p ro v ed n o n -R I M A P R E C test

sh o w s its u n d e n ia b le ad v an tag es o v er rctAŨ

test: m uch m o re co st-effec tiv e and less tim e-

consum in g T h e re íò re , it is high ly

reco m m en d ed th a t th is test sh o u ld be u sed as an

altem a tiv e to rc /4 0 test for in vitro exam in ation

o f p o lio m y elitis vaccine

4 D iscu ssio n

A n u m b e r o f stud ies have estab lish ed that

ch ang es in th e n u cleo tid e seq u en ces o f vaccine

viruses can lead to the d ev elo p m en t o f

n eu ro v iru len t rev ertan ts [8,9] F in ding s from

stu d ies o f ty p e 3 v iru s p ro v id e so un d evidence

that a single n u cleo tid e ch an g e in the b ase at

position 4 7 2 in th e geno m e co rrelates directly

w ith in creased n eu ro v iru len ce for m onkey

[6,9] O ur re su lt d em o n strates th at the im proved

non R I-M A P R E C test is u sefu l for in vitro

assessm ent o f th e safety o f sin g le h arvests used

to prod uce triv a le n t O PV

D esp ite m a n y ad v an tag es o f no n RI-

M A P R E C o v e r R I-M A P R E C as listed above,

the d etectab le ra n g e o f n o n -R I M A P R E C is

narrow er th an th a t achiev ed b y M A P R E C : in

estim atin g th e c o n te n t o f b ases ch an g es b y non-

RI M A P R E C , fo r all th ree ty p es o f virus the

u p per lim it is 1 5-20% (b ecau se th e ílu o rescen ce

intensity o f d ig e ste d ban ds is to o strong to be

m e a s u rỉd ) and th e lo w est lim it is 0.3-0.4% (b ecause th e intensity is too thin to be

m easu red ) T h ese com p arative results

d em o n strate th at M A P R E C h as the ađvantage

o f b ein g a b le to d etect a w id er rang e o f changes

in n u cleo tid e seq u en ces than no n-R I M A PR E C

H o w ever, since the q u an tity o f n ucleotide change sho w n to co n fer n eu ro virulent

p ro p erties to O P V is w ith in th e rang e o f

d etecta b ility o f n o n -R I M A P R E C , it is

su g g ested th at e ith er p ro ced u re m ay be used to test O P V for n eu ro v iru len t p ro perties [2] Indeeđ, o n th e b a sis o f practical con ditions in

o u r lab o rato ry , th e im p ro v ed non R I-M A P R E C

te st is re a lly o f g reat adv an tag e

T h e rc /4 0 te st is ữ e q u e n tly used in our lab o rato ry to assess th e v iru len ce o f the vaccine viru ses v ia th e ir tem p eratu re sensitivity The rev ertan t is n o t tem p eratu re sensitive, thereío re

th e ir rep ro d u ctiv e cap a city has n o chan ges at the te m p eratu res o f 3 6 ° c an d 4 0 ° c T hus, by

e x am in in g the rep ro d u ctiv e cap acity o f vaccine viru ses at d ifferen t tem p eratu res an d com paring these resu lts, w e can in d irectly assess the genetic m aterial o f the vaccin e H ow ever,

n u cleo tid e m u tatio n s th a t have the greatest

co n trib u tio n to v iru len ce have on ly m inor e íĩe c t

on viral te m p eratu re sen sitivity T herefo re, if a vaccine sam p le sh o w s tem p eratu re sensitivity,

it does n o t m ean to tally th at th is sam ple is not

n eu ro v iru len t, w h ich is a g reat disad vantage o f

rct40 te st th at can b e overcom e b y M A PR E C

M o reo v er, co m p ared in term s o f tim e consum ed

an d ex p en ses, the im p rov ed non R I-M A P R E C test sh o w s its u n d en iab le adv an tag es ov er rct40 test

T h ese resu lts, th e re íb re , d em on strate that

th e im p ro v ed n o n R I-M A P R E C test should be

u sed as a n a ltem a tiv e to the usual rctAO test

w h ich h a s th e d isad v an tag es o f being

u n av o id ab ly co stly an d tim e-co n su m in g , to

su p p lem en t the M N V T G en o m ic m onitoring

150 T.T.H A n h , L.T Luan i V N U Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 145-151

by this m etho d sh o u ld p ro v e to b e im p o rtan t in

o rd er to co n firm th e co n siste n cy o f vaccine

viru ses during pro d u ctio n

A ck n o v v led g em en ts

W e are gratefiil to o u r colleag u es in the Q C

departm en t (P o liovac) an d D r N g u y en D ang

H ien -d irecto r o f P o lio vac fo r th e ir great

support; D r D ang T u a n D at-V accin e and

B iological P roducts co m p an y N o l, D r N gu yen

V an M ui and N gu yen Q u an g H u y o f C o llege o f

Science, V N U as w ell as m an y scien tists at

Japan P o lio m y elitis R esearch Institu te for th eir

great h elp o f n ece ssary ch em ica ls and

equipm ent; and also, w e th a n k D r P ham V an

T y o f C o lleg e o f S cience fo r h is inv alu able

com m ents

R e íe re n c e s

[1] K.M C hum akov, M ulant anaỉysis by PCR and

restriction enzyme cleavage (M A PR EC ) for oral

poliovirus (Sabin) vaccine, Standard operating

proccdure, prcpared for the W H O collaborative

study on M APREC, USA Version 3.2 January

2 5 (1 9 9 9 )

[2] Hitoshi Horie, Y oshio Tano, Y utaka Doi, So

Hashizume Estimation o f the neurovirulence o f

poìiovirus by non - radioisotopc m olccular

analysis to quantiíy genom ic changes

Biologicals, USA.26 (1998) 289.

[3] B.M, Nkowane, S.G.F \Vassilak, W.A Orenstein, K.J Bart, L.B Schonbergcr, A R Hinm an, O.M Kew, V accine - associated paralytic poliom yelitis, U nited States: 1973 through 1984,

JAM A, USA 257(1987)1335.

[4] A drian M cG oldrick, J A ndrew M acadam ,

G lynis D unn, Alison Rowe, John Burlison, D Philip M inor, Janet M eredith, J David Evans, and Jeffrey W A lm ond R olc o f m utations G-480 and C-6203 in the attenuation phenotype o f

Sabin type 1 Poliovirus J Virol, USA 69 (1995)

7601

[5] D Philip M inor, Poliovirus vaccination: current understanding o f poliovirus interactions in hum ans and im plications for the crađication o f

poliom yelitis, Exp Rev M oỉ M ed Online, UK

Septcm bcr 23, 1999

[6] Saum itra Das, M ichael Ott, Akemi Yamann,

A run V enkatesan, Sanjecv Gupta, A sim

D a sg u p ta , Inhibition o f intem al entry site (IR E S)-m cdiated translation by a small yeast RNA: a novel strategy to block hepatitis c virus

protcin synthesis, F ro n tie rs in B ioscience,

U S A 3(1 9 9 8 ) 1241 [7] M aria-M agdalena, G eorgescu, M aryse Tardy- Panit, Sophie G uíllot, Radu C rainic and Francis

D clpeyroux, M apping o f m utations contnbuting

to th e tem perature sensitivity o f the Sabin 1

vaccine strain o f poliovirus J.Virol, USA 69

(19 9 5 )5 2 7 8

[8] K aw am ura N , K ohara M, A be s et aỉ.y

D cterm inants in the 5’ non coding region of poliovirus Sabin 1 RNA that influence the

attenuation phenotypc, J Viroỉ, USA 63 (1989)

1302

[9] G D W estrop, K.A W archam , D.M A Davis et

a i, G enetic basis o f attcnuation o f the Sabin type 3 oral poliovirus vaccine, J Virol, USA 163

(1989) 1338

T.T.H A n h , L.T Luan / VNƯ Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 145-151 151

Sử dụng kỹ thuật Non-Ri Maprec cải bỉên đánh giá sự an toàn của Vaccine bại liệt uống giảm độc lực (OPV)-iru thế so với

Thiều Thị Hoài Anh, Lê Thị Luân

Trung tâm khoa học sản xuất vaccine Sabin (Poliovac), 135 Lò Đúc, Hà Nội

K ỹ th u ậ t M A P R E C - p hân tích đ ộ t biến bằng kỹ th u ậ t P C R và p h ân cắt bằn g en zy m e giới hạn (M u tan t an aly sis by p o ly m erase ch ain reactio n and restric tio n enzym e cleav ag e) sừ dụng đồng vị phóng x ạ 32p lần đ ầu tiên đ ư ợ c đ ư a ra bởi C hu m ako v và cộng s ự đã tỏ ra rất hữ u hiệu tro n g việc đánh giá vật c h ấ t di tru y ền của v iru s bại liệt, cụ thể là n h ữ ng th ay đổi tro ng cấu trú c geno m e có thể dẫn đến tính độc cù a v a c c in e bại liệt số n g u ố n g giảm độc lực (O P V ) S au đó, kỹ th u ật M A P R E C không sử

dụ n g đ ồ n g vị p h ó n g xạ (non R I-M A P R E C ) đã đư ợc phát triển d ự a trên kỹ th u ậ t M A P R E C c ơ bản nói trên bời các nhà k h o a họ c ở V iện n g h iên cứ u bại liệt N h ật Bản v à o năm 1998 và đã tỏ rõ m ột số ưu thế

so với M A P R E C , g iú p ch o v iệc ứ n g d ụ n g ở các nư ớc đ an g p h át triển n hư nư ớc ta đư ợc dễ dàng hơn Tại phòng thí n g h iệ m củ a ch ú n g tô i, kỹ th u ậ t non R I-M A P R E C tiếp tục đ ư ợ c cải biến để phù hợp hơn với điều kiện cụ th ể cùa V iệt N am , đ ồ n g thời ch ú n g tôi cũng so sánh để nêu ra n hữ ng ưu điểm to lớn cùa kỹ th u ậ t cải b iến này với m ộ t th í nghiệm "cổ đ iể n ” vẫn đ ư ợ c sử d ụ n g tro n g p h ò n g thí nghiệm để đánh g iá sự an to à n cùa sản phẩm v acc in e-th ừ nghiệm nhiệt độ rct40