VNU Jo u m al of Science, N atu ral Sciences and T echnology 23 (2007) 145-151 ưsing improved Non Ri -Maprec assay to detect virulence mutations in poliomyelitis Vaccine viruses: advantages over rctA test Thieu Thi Hoai Anh*, Le Thi Luan Poliomyelitis vaccine Research and Production center (Poliovac), 135 Lo Duc, Hanoi, Vietnam R eceived 23 January 2006 A b s tra c ỉ M utant analysis by polym erase Chain reaction an d restrictio n enzym e cleavage (M A P R E C ) has been d ev e lo p c d b y C hum akov et aỉ for p o lio v iru s to determ in e qu an titativ ely the p resence o f g enom ic changes in particu lar nucleotide sequences th at lead to v u len ce phenotypes o f oral p o lio m y elitis vaccine (O P V o r live attenuated v ac cin e) [1] A fterw ards, this RI (rad io iso to p e) M A P R E C w as d eveloped to non RI - M A P R E C b y Jap an P oliom yelitis R esearch Institute in 1998 to com pensate for som e disadvantages o f R I-M A P R E C [2] T h is test w as highly recom m endod for laboratories to assess the saíety o f their v ac cin e P roducts A t P oliovac, the rctA0 test w as u sed to be considered a m ajor tool for vaccine pro d u cts exam in atio n In this research, we h ave im p ro v ed the non RI M A P R E C test to apply it w ell w ith p ractical co n d itio n s in our laboratory and com pared this test to the usual rct40 test to point o u t its g re at advantages In tro d u c tio n activity T h u s strict control is strongly required during v accin e p roduction P oliom yelitis is an acute d isease o f the C entral nervous sy stem (C N S ) caused by poliovirus O ral p o lio m y elitis vaccin e (O P V ) is usually co n sid ered the m ajo r tool for eradication o f p o lio m y elitis in m any countries, as well as V ietnam It is reg ard ed as one o f the safest vaccines in cu rren t use, but som e polio vaccine related - p araly tic cases have been reported recently [3] T h ese cases are caused by vaccine viruses un d erg o in g rev erted m utations during repeatcd replications A lth o u g h th e re are m an y alterations betw een v iru len t and norm al íorm s o f polio v iru ses: for S ab in type 1, and 3, vaccine strains d iffe r fro m th eir p ro g en ito rs by 55, 23 and 11 m u tatio n s, resp ectiv ely [4], few changes required fo r a tten u aticn and reversion T hat is the reason w h y ty p e S abin strain has the least stable g en o m e an d thus can easily revert to virulent fo rm w h ile Sabin type 1, in contrast, h as the m o st stable genom e It w as dem onstrated that neurovirulence increased w hen the fo llo w in g changes to o k Dlace in base positions o f th e viral genom e: in serotype , p o sition in the non co d in g region (N C R ) changed fro m G to A, p o sitio n 525 changed írom u to c an d th e re ío re co m p en sated for the presence o f G -4 b y resto rin g base-p airing in P oliovirus has a g en o m e o f single-stranded RNA, its rate o f m u tations is usu ally high during replication sin ce R N A -d ep en d en t R N A polym erase does n o t h ave the p ro o f - reading * Corĩesponđing author Tel.: 84-4-8531213 E-mail: tthanh@vnmilk.com.vn 145 146 T.T.H Anh, L.T Luan / V N U Ịoum aỉ of Science, Natural Sciences and Technology 23 (2007) 145-151 the stem -loop structure [5]; for serotype 2, w hich are th reaten ed th e m o st by poliom yeli tis position 481 changed from A to G, prohibiting the íbrm ation o f a n ew b ase p air betw een residues 481 and 511 th a t w eakens the secondary structure [5]; for ty p e 3, p o sition 472 changed from u to c, resto rin g the polypyrim iđine tract-b in d in g p ro tein (an A íu rth er co n sid eratio n is that its use carries the initiation factor in n eu ro b lasto m a cells [ ]) site A ll these nucleotides lay w ith in the region designated “intem al rib o so m al en try site” (IR ES), o f w hich th e stability has a considerable in ílu en ce on th e translation efficiency o f v iru s-sp eciíĩc p ro tein s associated w ith neurovirulence, in o ther w ords, these m utations interfere w ith the IR ES ability to interact w ith /ra/w -acting factors C hum akov et al intro d u ced a m ethod designated “M utant analysis b y polym erase Chain reaction and restriction enzym e cleavage” (M A P R E C ) to estim ate the ratio o f viruses containing genes o f a v iru len t natu re in a vaccine virus population W h en the p roportions o f such genes ex ceed a particu lar level, the vaccine fails the m onkey neu ro v iru len ce test (M N V T): for type 1, app ro x im ately 5% o f 480A and 525-C com bined; for type 2, betw een 1.7% and 3.7% o f 481-G ; fo r type 3, about 1% o f 472-C A t present, M A P R E C can b e used as a supplem ent to the M N V T for several purposes: establishing and m on ito rin g o f m olecular consistency o f vaccine p roduction, as a prelim inary m olecu lar te st befo re M N V T , as a substitute o f the rct40 test, screening o f single harvests and m onitoring the genetic stability o f vaccine viruses T he non-R I M A P R E C test w as developed from R I-M A P R E C by Japan P oliom yelitis risk o f cau sin g env iro n m en tal problem s [2 ] T h e rct 40 test (rep ro đ u ctiv e capacity at ’C ) is used to be co n sid ered a sim ple tool for assessing th e viru len ce o f vaccine viruses via their tem p eratu re sensitivity T he rev ertan t is not tem p eratu re - sensitive, th ereío re its rep ro d u ctiv e cap acity h a s no difference at the 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 capacity o f vaccine viruses at th ese tem p eratu res and com paring the results, th e g enetic m aterial o f the vaccine can b e in d irectly assessed H ow ever, m u tations th a t have the g reatest co n trib u tio n to virulence h av e o nly m in o r effect on viral tem p eratu re sensitivity b u t o th er nu cleo tid es (6203, 7071, 7410 an d 7441 for type 1; 2034 for type 3) [4,5,7] T h ere ío re, i f a single h arvest show s tem perature sensitivity, it does not m ean totally this sam ple is n o t neurovirulent T h is g reat d isadvantage o f rc/4 test can be d eíin itely o vercom e b y M A P R E C In this study, w e p eríò rm ed im proved n on R I-M A P R E C assay on ty p e vaccine viruses, w hich h ave th e g reatest p o ssibility o f revertant m utation to assess th eir saíety and com pared this test to the usual rctAO test in term s o f effectiveness, ex actitude, tim e consum ed and expenses O n the b asis o f these results, we reco m m en d ed One o f th ese tw o tests for the in vitro ex am in atio n o f oral po lio v acc in e’s safety in o u r laboratory M a te r ia ls a n d m e th o d s R esearch Institute in 1998 to com pensate for the biggest disadvantage o f R I-M A PR E C : its 2.1 Viraỉ RNA extraction and cDNA synthesis use o f radioisotope req u ires a h igh level o f expertise and the use o f equ ip m en t, w hich, In th is stu d y w e u sed six type p o lio virus unlike in econom ically d ev elo p ed countries, single h arv ests p ro d u ced in 2005 at Poliovac m ay not be available in d ev elo p in g countries designated 402, 403, 404, 405, 406, 407 and T.T.H Anh, L.T Luan / VN U loum al o f Science, Natural Sciences and Technology 23 (2007) 145-151 147 P oliom yelitis 2.2 MAPREC, non Rl-MAPREC and improved non RỊ-MAPREC Positive single-stran d ed R N A o f poliovirus B oth M A P R E C an d non R I-M A P R E C are quite identical except for the w ay these two m arker sam ples designated 472-a, -b , 472-c, 472-d provideđ by Japan R esearch Institute w as extracted from 0.5 ml virus su sp en sio n by phenol extraction w ith sodium d o d ecy l sulfate (S D S ) o f final cen tratio n o f 0.5 % T he viral R N A w as p recipitated using 100% isopropanol and treated w ith ethanol 70% , cD N A w as then synthesized w ith M oloney m u rin e leukem ia virus reverse Inviừ ogen), tran scrip tase ran d o m (M M L V -R T h ex an u cleo tid e - prim ers (F erm entas) and incubated for lh at 37"C m ethods u sin g to q uantify d igested D N A bands F o r p o lio v iru s type 3, a v irulent base c at resid u e w as exam in ed in our research Since n o k n o w n restrictio n enzym e cuts either atten u ated or rev erted po lio v iru s sequences at nucleotide 47 , a restrictio n site o f Mbol is en g in eered b y P C R usin g a m utagenic prim er T h u s the restrictio n site is com posed, in part, o f the p rim er sequence and in part, o f the viral cD N A u sed as a tem plate T ab le P C R prim ers u sed to detect m utations b y M A P R E C and non R I-M A P R E C ♦MAPREC P rim er p o larity sense an tisense P rim er seq u en ce T 1G A G C T A C A T G A G A G T gC T C C G G C C C C T G A A T G C G G C T gA 470 C 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 m o d iíĩcatio n s A g at p o sitio n 447 w as introduced to destroy com pared to the po lio v iru s seq u en ce w hich restriction site for Hinữ site w hile a g at w ere introduced to create o r d estro y restriction po sition w as inserted to create restriction sites T he norm al vaccine sequence w as cut by sites for b o th Hi nữ and Mbol L ow ercase letters show Hinĩl, the revertant sequence w as cu t b y Mbo I ♦ n o n R I-M A P R E C P rím er p o larity P rim er sequence _ sense T440G A G A G TC C TC C G G C C C C TG A A TG C G G C TgA T471 an tisense A 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 502 n lik e M A P R E C , in non R I-M A P R E C , only Mboỉ w as u sed to cu t th e revertant sequence PC R am p liíĩcatio n usin g sense and antisense prim ers as describ ed p rev io u sly and Taq D N A po ly m erase (In v iừ o g en , PerkinElm er) w as co nducted for 40 cycles F o r R I-M A P R E C , PC R am p liíicatio n w as asym m etric (i.e a -fold ex cess o f sense prim er ensures that the p red o m in an t p roduct o f this reaction is sin g le-stran d ed D N A : 30|Jg/m l for sense prim er an d ng/m l fo r an tiserse prim er) T h e second strand is synthesized using a 32 p lab eled an tisen se prim er A fter treatm ent o f the am p liíie d D N A p roduct w ith Mboỉ, the d igested m aterial w as separated by elecừ o p h o resis in polyacry lam id e gel The m utatio n al change (percentage o f 472C ) w as calculated by m easu rin g radioactivity in counts p er m in u te (cpm ) o f d igested an d undigested bands, u sin g the equation: d ig ested D N A band (cpm ) o v er d igested D N A b an d (cpm ) plus u n d ig ested D N A b an d (cpm ) 148 T.T.H Anh, L.T Luan / V N U Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 145-151 F or non R I-M A P R E C , P C R am p liíĩcatio n w as perform ed using the p rim ers show n in T able 1, w ith sense an d an tisen se prim ers at equim olarity (both at a íinal co ncentration o f 3ụg/m l) to iíave the PC R pro d u ct o f 93bp A fter treatm ent o f 25^1 o f the D N A p ro d u ct w ith the com position of Mboỉ and its buffer (Ferm entas), the digested m aterial consisting o f D N A bands o f 30bp and 63bp w as applied to a 12% polyacrylam ide gel to g eth er w ith X loading buffer, and electro p h o resed w ith IX T A E buffer o r 0.5X T B E b u ffer for 2h at room tem perature, using 100V A fìerw ard s, the gel w as stained for 30m in w ith SY B R G reen (Sigm a) điluted 10000 -fo ld w ith T A E o r TB E T he digesíed D N A b an d s w ere d etecteđ by irradiating w ith u v rays at a w avelength o f 250nm and their qu an tities w ere d eterm ined by the use o f a dual-w avelength chrom atoscanner T ogether w ith groups o f reco m b in an t m arker viruses designated 472-a, 472-b, 472-c, 472-d, for w hich the percentage o f 72-C values (determ ined by R I-M A P R E C ) w ere 0.72% , 1%, 2.8% and 4.0% , respectively, the calibration curve w as prepared, in w h ich the axis o f abscissa indicates the A uorescence intensity o f digested D N A bands and the axis o f ordinate indicates the percentage q u an tities o f 472-C s in g this calibration curve, w e can determ ine the 472-C percentage o f each single h arvest based on th e A uorescence in ten sity o f digested D N A b an d s and d ecid e w h eth er it passes the M N V T o r not F or im p ro v ed non R I-M A P R E C applied in ou r lab o rato ry , w e co m p ared the electrophoresis p attem o f virus sam p les to th e m arker virus groups A sam ple p asses the test i f no 63bp and 30bp d ig ested D N A b an d s w o u ld ap p ear or they appear b u t the b an d s are w eak er than the c o e sp o n d in g b an d s o f -b m arker (472C p ercen tag e o f 1%) - like the p a tte m o f 472-a m arker (4 C p ercen tag e o f % ) For any case that is hard to co m p are b y u n aided eyes, the sam p les should be retested b y rcí 40 test R e s u lts Fig sh o w s the p attem obtained by electro p h o resis in an aly zin g ty p e polioviruses and g ro u p s o f reco m b in an t m a rk e r viruses A ll the v accin e sam ples p ro d u ced at Poliovac in 2005 sh o w n o 30bp an d b p b an đ s w hile the co rresp o n d in g bands o f 472-b, -c, 472-d are obvious T h e 63bp b an d o f -a is hard to d etect sin ce its p ro p o rtio n o f C is ju st 0.72% AU the type single h arv ests pass the non R I-M A P R E C and are safe fo r use Fig E lectrophoresis p a tte m o f type poliovirus b y non R I-M A P R E C w ith p B R 2 /A /jp I lad d er, arrows indicate 90bp, 67bp and 30bp bands , , , , 5, 6, are vim s sam p les (single harv ests) d esig n ated 402, 403, 4 ,4 ,4 , 407, respectively 8, 9, 10, 11 are 472-d, 472-c, -b and 472-a, resp ectiv ely 149 T.T.H A nh, L Ĩ Luan Ị V N U Ịournal o f Science, Natural Sciences and Technology 23 (2007) 145-Ĩ5Ĩ It th e re ío re can be affirm ed that M A P R E C test is b o th effectiv e and exact We also m e a su rỉd ) and th e low est lim it is 0.3-0.4% (because the intensity is too thin to be com pared this test to the usual rctAỒ test in m easured) term s o f tim e co n su m ed and expenses d em onstrate that M A P R E C h as the ađvantage T h ese com parative results o f b ein g ab le to detect a w id er range o f changes T able2 C o m p ariso n b etw een the tw o tests in term s o f tim e co n su m ed and expenses (p e r sam ple) T im e co n su m ed E xpenses 98h -4 U S D rct4 test -1 Ư S D n o n R I-M A P R E C test 25h So, co m p ared in term s o f tim e consum ed in nucleo tid e sequences than non-R I M A PR EC H ow ever, change since the quantity o f nucleotide show n to co n fer neurovirulent p ro p erties to detectab ility O PV of is w ithin the range o f n on-R I M APREC, it is suggested that eith er p ro ced u re m ay be used to and expenses, im proved n o n -R I M A P R E C test test O PV for n eu ro v iru len t properties [2] show s its u n d en iab le ad v an tag es ov er rctAŨ Indeeđ, o n th e b asis o f practical conditions in test: m uch m o re co st-effectiv e and less tim econsum ing T h ere íò re, it is highly o u r laboratory, the im proved non R I-M A PR EC recom m ended th a t this test should be used as an T h e rc /4 test is ữ eq u en tly used in our altem ative to rc /4 test for in vitro exam ination laboratory to assess the virulence o f the vaccine o f p o liom yelitis vaccine viruses v ia th eir tem p eratu re sensitivity The test is really o f great advantage revertant is not tem perature sensitive, thereíore th eir rep ro d u ctiv e cap acity has no changes at D iscussion the tem p eratu res o f ° c and °c Thus, by ex am in in g the rep ro d u ctiv e capacity o f vaccine A nu m b er o f studies have estab lish ed that viruses at different tem p eratu res and com paring changes in the n u cleo tid e sequences o f vaccine these resu lts, viruses genetic can lead to the dev elo p m en t of w e can m aterial indirectly assess the o f the vaccine H ow ever, neurovirulent rev ertan ts [8,9] F indings from n u cleo tid e m u tatio n s th at have the greatest studies o f type virus p rovide sound evidence co n tribution to viru len ce have only m inor eíĩect that a single n u cleo tid e change in the base at on viral tem p eratu re sensitivity T herefore, if a position 472 in th e genom e correlates directly vaccine sam p le show s tem perature sensitivity, w ith m onkey it does n o t m ean to tally that this sam ple is not [6,9] O ur resu lt dem onstrates that the im proved n eu ro v iru len t, w h ich is a great disadvantage o f non R I-M A P R E C test is useful for in vitro rct40 test that can b e overcom e b y M A PREC assessm ent o f th e safety o f sin g le harvests used M o reo v er, co m p ared in term s o f tim e consum ed to produce triv alen t O PV and expenses, the im proved non R I-M A PR EC increased D espite neu ro v iru len ce m an y ad vantages for of non RI- M A PR E C o v er R I-M A P R E C as listed above, test sh o w s its u n d en iab le advantages over rct40 test the detectable ran g e o f n on-R I M A P R E C is T h ese resu lts, th ereíb re, dem onstrate that narrow er than that achieved b y M A P R E C : in th e im p ro v ed n o n R I-M A P R E C test should be estim ating the co n ten t o f b ases changes b y non- used as an altem ativ e to the usual rctAO test RI M A P R E C , fo r all th ree types o f virus the w hich upper lim it is 15-20% (because th e íluorescence u n av o id ab ly co stly intensity o f d ig ested bands is to o strong to be su p p lem en t the M N V T G enom ic m onitoring has th e d isad v an tag es and of being tim e-consum ing, to 150 T.T.H A nh, L.T Luan i V N U Ịoum al o f Science, Natural Sciences and Technology 23 (2007) 145-151 by this m ethod should p ro v e to be im portant in order to confirm the co n sisten cy o f vaccine viruses during production A cknovvledgem ents W e are gratefiil to o u r colleagues in the Q C departm ent (P oliovac) and D r N guyen D ang H ien-director support; Dr of P oliovac D ang T u an for th eir great D at-V accin e and B iological Products co m p an y N o l, D r N guyen V an M ui and N guyen Q u an g H uy o f C ollege o f Science, V N U as w ell as m any scientists at Japan Poliom yelitis R esearch Institute for their great help of n ecessary ch em icals and equipm ent; and also, w e th an k Dr P ham V an T y o f C ollege o f Science for his invaluable com m ents R e íe re n c e s [1] K.M Chum 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 HO collaborative study on M APREC, USA Version 3.2 January (1 9 ) [2] Hitoshi Horie, Yoshio Tano, Yutaka Doi, So Hashizume Estimation o f the neurovirulence o f piovirus by non analysis to quantiíy radioisotopc m olccular genom ic Biologicals, USA.26 (1998) 289 changes [3] B.M, Nkowane, S.G.F \Vassilak, W.A Orenstein, K.J Bart, L.B Schonbergcr, A.R Hinman, O.M Kew, Vaccine associated paralytic poliom yelitis, U nited States: 1973 through 1984, JAM A, USA 257(1987)1335 [4] Adrian M cGoldrick, J Andrew M acadam, G lynis Dunn, Alison Rowe, John Burlison, D Philip M inor, Janet M eredith, J David Evans, and Jeffrey W Alm ond R olc o f m utations G-480 and C-6203 in the attenuation phenotype o f Sabin type 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 Moỉ M ed Online, UK Septcm bcr 23, 1999 [6] Saum itra Das, Michael Ott, Akemi Yamann, A run Venkatesan, Sanjecv Gupta, A sim D asgupta, Inhibition o f intem al entry site (IRES)-m cdiated translation by a small yeast RNA: a novel strategy to block hepatitis c virus protcin synthesis, F ro n tiers in B ioscience, U S A 3(1998) 1241 [7] M aria-M agdalena, Georgescu, M aryse TardyPanit, Sophie Guíllot, Radu Crainic and Francis D clpeyroux, M apping o f m utations contnbuting to the tem perature sensitivity o f the Sabin vaccine strain o f poliovirus J.Virol, USA 69 (1995)5278 [8] K awam ura N, Kohara M, Abe s et aỉ.y D cterm inants in the 5’ non coding region of poliovirus Sabin 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, Genetic basis o f attcnuation o f the Sabin type 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 an toàn Vaccine bại liệt uống giảm độc lực (OPV)-iru so với thử nghiệm nhiệt độ r c t4 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 ật M A P R E C - phân tích đột biến kỹ th u ật P C R phân cắt enzym e giới hạn (M utant analysis by p o ly m erase ch ain reaction and restrictio n enzym e cleavage) sừ dụng đồng vị phóng x 32p lần đ ầu tiên đư ợ c đ a C hum akov cộng tỏ hữu hiệu việc đánh giá vật ch ất di truy ền virus bại liệt, cụ thể nhữ ng thay đổi cấu trúc genom e dẫn đến tính độc cùa v accin e bại liệt sống u ố n g giảm độc lực (O PV ) Sau đó, kỹ thuật M A PR E C khơng sử dụng đ ồng vị p h ó n g xạ (non R I-M A P R E C ) phát triển d ự a kỹ th u ật M A PR E C nói bời nhà k h o a học V iện nghiên cứu bại liệt N h ật Bản v năm 1998 tỏ rõ m ột số ưu so với M A P R E C , g iúp ch o việc ứ ng d ụng nước p h át triển nước ta dễ dàng Tại phòng thí n g h iệm ch ú n g tôi, kỹ th u ật non R I-M A P R E C tiếp tục đư ợc cải biến để phù hợp với điều kiện cụ th ể cùa V iệt N am , đ n g thời so sánh để nêu nhữ ng ưu điểm to lớn cùa kỹ thuật cải biến với m ộ t thí nghiệm "cổ đ iển ” đ ợ c sử d ụ n g p hòng thí nghiệm để đánh giá an to àn cùa sản phẩm vaccin e-th nghiệm nhiệt độ rct 40 ... altem ative to rc /4 test for in vitro exam ination laboratory to assess the virulence o f the vaccine o f p o liom yelitis vaccine viruses v ia th eir tem p eratu re sensitivity The test is really... hus, by ex am in in g th e rep ro d u ctiv e capacity o f vaccine viruses at th ese tem p eratu res and com paring the results, th e g enetic m aterial o f the vaccine can b e in d irectly assessed... p ro d u ced in 2005 at Poliovac m ay not be available in d ev elo p in g countries designated 402 , 403 , 404 , 405 , 406 , 407 and T.T.H Anh, L.T Luan / VN U loum al o f Science, Natural Sciences