SINTEZA LUCRARII FAZA 2007
Director de proiect Dr. Anda Baicus
PROIECT Nr. 219
“Genotiparea tulpinilor de poliovirus izolate in Romania in perioada 2007-2010 de la cazurile cu PAF si contactii sanatosi ai acestora ca obiectiv major al procesului de Eradicare a Poliomielitei”
În mai 1988 cea de-a 41 Adunare Mondială a Sănătăţii a propus Statelor membre ale OMS Eradicarea Globală a Poliomielitei până în anul 2000. Decizia eradicării a fost urmarea succesului enorm al vaccinării (mai ales cu vaccin polio oral VPO). VPO elimină virusul sălbatic din circulaţie (imunitate de grup) pentru că induce şi imunitate locală la poarta de intrare (IgA secretor). VPI (vaccin polio inactivat) protejează foarte bine individul, dar mai puţin colectivitatea. Dezavantajul utilizării VPO îl reprezintă riscul apariţiei cazurilor de poliomielită paralitică asociată vaccinării (VAPP). În 1989 a fost aprobat Planul de Acţiune pentru eradicarea poliomielitei. In 2003 a fost adoptat Planul Strategic de continuare a Programului de Eradicare Globală a poliomielitei pentru 2004-2008 .
a) Acoperire vaccinala ridicată :
· Obţinerea şi menţinerea unei acoperiri vaccinale corespunzătoare cu cel putin trei doze de vaccin polio oral trivalent (VPOT) în primul an de viata (la noi prin vaccinarea de rutină este raportată o rată de acoperire vaccinală cu 4 doze VPOT în primul an de viaţă de peste 90%).
· Vaccinare suplimentară (în ţări cu poliomielită endemică) prin organizarea zilelor naţionale de vaccinare (la noi deşi ultimul caz de poliomielită cu poliovirus sălbatic s-a înregistrat în 1992, în 1996 s-au organizat zile naţionale de imunizare (NIDS ) la toţi copiii sub 6 ani, care au primit câte 2 doze de VPOT, indiferent de numărul dozelor primite anterior şi fără a interfera cu vaccinarea de rutină).
· Vaccinare mop-up/ratissage (din poartă în poartă) – în ţări aflate în faza finală a întreruperii transmiterii poliovirusurilor sălbatice, constă în administrarea VPOT din casă în casă, tuturor copiilor din zonele cu risc mare (în special acolo unde acoperirea vaccinală este deficitară).
b) Supravegherea cazurilor de paralizie acută flască (PAF)
- Depistare, investigare de laborator, raportare a tuturor cazurilor de PAF survenite la copii mai mici de 15 ani indiferent de cauză. Prin definiţie, în acest sistem de supraveghere se includ şi toate cazurile de sindrom Guillain-Barre, mielită transversă, ca şi alte îmbolnăviri care produc PAF. Frecvenţa medie a acestora este de 1 caz la 100 000 indivizi sub 15 ani. O supraveghere corectă a cazurilor de PAF trebuie să evidenţieze toate aceste cazuri (1/150 000 sub 15 ani).
- Investigarea contacţilor sănătoşi ai cazurilor PAF (5 contacţi /1 caz PAF).
În 1988 răspândită încă epidemic pe toate cele 5 continente, poliomielita provocată de poliovirusurile sălbatice mai este găsită acum numai în părţi din Africa şi Sudul Asiei.
Progresele făcute din 2000, 2001, 2003, 2006 includ reducerea numărului de ţări polio-endemice de la 20 la 10, 6, 4. Transmisia poliovirusului a fost stopată în ambele Americi şi Pacificul de Vest. Europa a fost certificată ca “polio free” la reuniunea OMS/EURO din 5 iunie 2002. Riscul major al utilizarii VPO rămâne apariţia cazurilor de poliomielită paralitică asociată vaccinarii (VAPP) şi emergenţa şi circulaţia tulpinilor de poliovirus derivate din vaccin (VDPV). Pentru ca o tulpină Sabin să fie denumită VDPV trebuie să fi acumulat peste 1% mutaţii la nivelul genelor care codifică proteina capsidală VP1.
OBIECTIVE REALIZATE PENTRU FAZA ANULUI 2007
PCR-RFLP va fi aplicata tuturor tulpinilor de poliovirus izolate
PCR este o metodă rapidă de amplificare enzimatică a unui fragment specific de ADN. Amplificarea enzimatică in vitro reprezintă metoda prin care se sintetizează un număr mare de copii ale unui segment dintr-o moleculă de ADN. Un anumit segment de acid nucleic este replicat în mod specific pornind de la doi primeri oligonucleotidici care flanchează fragmentul respectiv de ADN. Procesul are loc în cadrul unor cicluri repetitive de operaţiuni care cuprind: denaturarea termică a ADN, hibridizarea primerilor, elongarea acestora cu ajutorul unei ADN polimeraze termostabile (Taq. polimeraza) folosind o matriţă ADN. Primerii, adăugaţi în exces faţă de ADN ce trebuie amplificat, hibridizează pe catenele ADN pe baza complementarităţii de secvenţă, câte un primer pe fiecare lanţ. Ciclurile succesive de amplificare vor dubla de fiecare dată cantitatea de ADN sintetizată în ciclul precedent, rezultând teoretic după 30 de cicluri o amplificare de aproximativ 270 milioane de ori a unui segment de ADN.
Evaluarea variaţiei genetice a tulpinilor de poliovirus izolate, va fi realizată prin analiza polimorfismului segmentelor de ADN provenite dintr-o regiune a genomului (RFLP), ce urmează revers transcrierii (J. Balanant şi col, 1991).
Principiul metodei constă în evidenţierea unui situs de restricţie care a fost creat sau a fost suprimat după apariţia unei mutaţii prin comparaţie cu numărul de situsuri corespunzătoare de restricţie ale tulpinilor vaccinale Sabin. Enzimele de restricţie sunt endonucleaze de origine bacteriană ce au proprietatea de a tăia ADN dublu catenar la nivelul unor situsuri specifice. Produşii de digestie se vizualizează în gel de agaroză 3% şi se evaluează in raport cu un marker de masă moleculară şi o probă de ADN nelizat alături de lizate ale tulpinilor de referinţă. Practic după amplificare şi digestie enzimatică, migrarea în gel de agaroză permite punerea în evidenţă a unei benzi suplimentare sau a absenţei altei benzi prin comparaţie cu tulpina vaccinală. Profilurile de restricţie ale tulpinilor vaccinale Sabin sunt conservate de-a lungul pasajelor inter- şi intraumane.
O tehnică « RT-PCR-RFLP new » pusă la punct în cadrul Institutului Pasteur Paris prevede examinarea a trei porţiuni din genom :
¨ regiunea VP3-VP1 utilizând amorsele UG24-UC1
¨ regiunea VP1-2A utilizând amorsele UG19-UC13
¨ regiunea 3D –3’ utilizând amorsele UG17-UC10
În cadrul acestei tehnici suspensia virală utilizată pentru obţinerea ADNc este diluată 1/10 (S. Guillot şi col, 2000).Protocolul de lucru utilizat pentru realizarea proiectul va fi cel elaborat de cercetători din Institutul Pasteur Paris (S. Guillot şi col, 2000) dar va fi optimizata in laboratorul nostru (vor fi testate si alte variante ale concentratiei primerilor si reactivilor)
Tehnica : ARN viral va fi revers transcris şi ADN complementar va fi amplificat prin PCR. După terminarea programului pe termociclator produşii de amplificare se vor identifica prin electroforeză în tampon TAE 1X, pe un gel de 1,5% agaroză (colorat cu bromură de ethidiu) şi se vizualizeaza cu ajutorul unui transiluminator cu lumină UV. Sunt investigate în acelaşi mod în paralel şi tulpinile vaccinale Sabin. Enzimele utilizate pentru digestie sunt trei endonucleaze: RsaI, DdeI, Hinf I. Un amestec de 21 ml realizat incubeaza 2 ore la 37 °C. Produşii RFLP se evidenţiaza prin electroforeză în gel de agaroză 3%, în tampon TAE 1X şi se vizualizeaza pe transiluminator în lumina UV. Profilurile obţinute în urma digestiei enzimatice sunt comparate cu cele obţinute pentru tulpinile vaccinale.
Secventele nucleotidice ale primerilor
UG24: (1913)-GACACCATGATTCCCCTTAA-(1932)
UC1: (2881)-GAATTCCATGTCAAATCTAGA-(2862)
UG19: (2870)-GACATGGAATTCACCTTTGTGG-(2891)
UC13: (3648)-TAGTACTTAGCTTCCATGTA-(3629)
UG17: (6536)-TCAGTGGCCATGAGAATGGC-(6555)
UC10: (7464)-TTTTTTTTTTTTTTTTTTTTTTTC-(7441)
1.2.2. Determinarea secventei acizilor nucleici in regiunea VP1-2A va fi aplicata pentru tulpinile de poliovirus la care situsuri de restricţie au fost create sau suprimate după apariţia uneia sau mai multor mutaţii prin comparaţie cu numărul de situsuri corespunzătoare de restricţie ale tulpinilor vaccinale Sabin. Secventierea altor regiuni ale genomului se va face numai daca profilurile de restrictie in regiunea 3D polimeraza o impun.
Tehnica : Secvenţierea nucleotidică pe un secvenţiator automat (ex. 3100 Avant – Applied Biosystems) se realizeaza in capilare care contin polimer de rezolutie foarte inalta – de tip POP 4 sau POP 6. Principiul constă în migrarea prin capilar a ADN marcat cu 4 fluorocromi diferiţi. Fragmentele de ADN sînt separate în funcţie de mărime şi traversează regiunea din dreptul unui fascicul laser. Laserul excită fluorocromii care emit fiecare un semnal cu lungimi de undă diferite, detectat de o cameră video cuplată cu un spectrograf (CDD). Prin intermediul unor filtre, datele privind intensitatea emisiilor sînt colectate şi conservate sub forma unor semnale electrice. Un program informatic, conectat la secvenţiator, permite analiza automatizată şi convertirea directă a datelor în secvenţe nucleotidice. Secventele se obtin in format ABI (electroforegrame) si in format text. Acestea sunt ulterior analizate cu programe informatice cum sunt BioEdit, Clustal w, Mega. Metoda de secvenţiere folosită în prezent este cea enzimatică, dezvoltată de Sanger. Această metodă se bazează pe utilizarea unor nucleotide modificate, 2′,3′didezoxiribonucleotid trifosfaţi (ddNTP), care joacă rolul de terminatori de lanţ în timpul reacţiei de polimerizare enzimatică a ADN. Datorită acţiunii ADN polimerazelor, ddNTP-urile sunt încorporate în catena în curs de elongare a ADN aşa cum sunt utilizate dNTP-urile normale. Diferenţa constă în faptul că un ddNTP incorporate, marcat cu fluorocromi, declanşează, la un moment dat, blocarea elongării. Dezvoltarea PCR a permis descoperirea şi caracterizarea unor noi ADN polimeraze, cum este Taq polimeraza, care este foarte utilă şi în secvenţiere, în special pentru regiunile cu structuri secundare. Secvenţierea prin PCR, integrată în sistemul automat, neradioactiv (cu fluorocromi), are avantajul că permite atât secvenţierea ADN după clonare într-un vector (ex. fagul M13) ca în celelalte sisteme de secvenţiere dar şi direct, a produsului obţinut prin PCR, fără o clonare prealabilă.
Pentru secventierea produsilor PCR purificati este utilizat kitul de secvenţiere ciclică a ADN AmpliTaqÒ FS Big Dye TM Terminators (Applied Biosystems).
Secvenţele vor fi aliniate folosind programul Clustal W, versiunea 1.83. Scanarea secvenţelor şi alinierea acestora va fi făcută cu programul Fast A (Gen Bank DNA). Secvenţele transmise de secvenţiator în formă de text vor fi comparate cu electroforegramele corespunzătoare (pentru detectarea erorilor de citire şi transcriere ale aparatului).
Analiza filogenetică – alinierea multiplă a secvenţelor
Programul Clustal W (Thompson si col 1994) caută alinierile optime folosind toată lungimea secvenţelor, folosind perechi de secvenţe. Secvenţele sunt comparate două câte două, rezultatul fiind un scor de similaritate, care serveşte la realizarea unei dendrograme.
1.3. EXPERTIZA EPIDEMIOLOGICA
1.3.1. Confirmarea si clasificarea cazurilor de poliomielita
Definiţia unui caz confirmat ca poliomielită este: “boala acută cu paralizie acută flască, compatibilă cu manifestările clinice ale poliomielitei, la care după analiza neurologică efectuată la 60 de zile de la debut se constată un deficit motor rezidual iar analiza virologică a evidenţiat prezenţa poliovirusului” (Expanded Programme on Immunization,WHO, 1989). Rezultatele investigaţiilor virologice (izolare, identificare, diferenţiere intratipică prin metode imunologice şi/sau moleculare) determină caracterizarea tulpinilor de poliovirus izolate ca fiind sălbatice, Sabin-like, sau derivate din vaccin (VDPV) (WHO Manual 2004).
Cazurile de poliomielită confirmate se clasifică ulterior în:
- cazuri produse de poliovirusul sălbatic (indigene sau importate )
- cazuri asociate vaccinării (VAPP)
- cazuri de origine necunoscută
1.3.2. Clasificarea tulpinilor de poliovirus in functie de omologia secventelor in regiunea care codifica regiunea VP1
Tulpinile OPV-like (Sabin-like): > 99% omologie a secvenţelor genomului cu tulpinile Sabin vaccinale. Recipienţii imunocompetenţi normal excretă tulpinile pentru o perioadă de 3 până la 6 săptămâni.
Tulpini derivate din vaccin VDPV : ≤ 99% omologie a secvenţelor între tulpinile izolate din teren şi cele vaccinale. Numărul mutaţiilor apărute indică timpul de circulaţie în populaţie. Au fost descrise două categorii de astfel de tulpini: VDPV (de la) imunodeficient (iVDPV ) şi VDPV circulant (cVDPV).
Tulpinile VDPV circulante (cVDPV) responsabile de apariţia unor epidemii au fost prima dată depistate cu cVDPV de tip 1 in 2000-2001 în Insulele Caraibe (2 decese înregistrate) şi Insulele Filipine (2001), în Madagascar cu cVDPV de tip 2. Un studiu retrospectiv efectuat în Egipt a demonstrat circulaţia unor tulpini de cVDPV de tip 2 în intervalul 1980-1990. Punerea în evidenţă a emergenţei şi a circulaţiei acestor tulpini ridică probleme majore în cadrul procesului de Eradicare Globală a Poliomielitei.
|
Nr. crt
|
EPID NR.
|
Nr. lab.
|
Nume
|
Varsta
|
Diagnostic
|
Data ultimei
vaccinari
|
Tulpina polio izolata
|
|
1.
|
ROMIS07005C1
|
25
|
Vladeanu
Petru Ciprian
|
0.6
|
contact
sanatos
|
VPOT2 – 25.10.06
|
PV3
|
|
2.
|
ROMBH07009C4
|
52
|
Lingurar Marius
|
2.10
|
contact
sanatos
|
VPOT4 – 30.09.05
|
PV1+2, PV1
|
|
Date de inoculare si pasaj
|
|
25/1 RD I :5.02.07
ECP: 9.02.07
T:12.02.07 PV3
25/1 L20B I: 5.02.07
ECP: 8.02.07
T: 16.02.07 PV3
|
25/2RD I :5.02.07
ECP: 9.02.07
T:16.02.07 PV3
25/2 L20B I: 5.02.07
ECP: 8.02.07
T: 16.02.07 PV3
|
|
52/1 RD I :8.03.07
P: 15.03.07
ECP:19.03.07
T:9.04.07 PV2
52/1L20B I :8.03.07
P: 15.03.07
ECP:19.03.07
T:9.04.07 PV2
|
52/2 RD I :8.03.07
P: 15.03.07 negativ
52/1L20B I :8.03.07
P: 15.03.07
ECP:20.03.07
T:9.04.07 PV1
|
Tab.2. Comparatie intre numarul tulpinilor de poliovirusuri izolate in anul 2006 si cele izolate in anul 2007
|
Serotipul
|
2006
|
2007
|
|
Virus polio 1
|
7
|
2
|
|
Virus polio 2
|
17
|
1
|
|
Virus polio 3
|
5
|
2
|
|
Total
|
29
|
5
|
3. INTRETINEREA SI PREPARAREA SUSPENSIILOR CELULARE (LINIILE L20B, RD) PENTRU TESTARILE EFECTUATE IN FAZA 2007
Tab.3. Numarul pasajelor efectuate pentru testarile pe tuburi si prin micrometoda in placi cu 96 godeuri
|
Nr. pasaj / data
|
L20B
|
RD
|
|
19.10.2007
|
P 49
|
P135
|
|
24.10.2007
|
P50
|
P136
|
|
26.11.2007
|
P158
|
P151
|
|
30.11.2007
|
P159
|
P152
|
4. MULTIPLICAREA TULPINILOR DE POLIOVIRUSURI DE REFERINTA
Multiplicarea tulpinilor de poliovirusuri de referinta (Sabin 1, 2, 3) s-a efectuat pe ambele linii celulare utilizate in laborator (RD si L20B), in flacoane de cultura cu suprafata de 75cm2 .
Tehnica : se verifica cultura celulara pentru confluenta si lipsa contaminarii bacteriene
- se indeparteaza mediul de crestere
- se inoculeaza 1 ml de antigen
- se lasa placa la adsorbtie, 20 min. la temperatura camerei
- se adauga mediu de intretinere (mediu Eagle MEM cu 2% ser fetal de vitel)
- placile se incubeaza la 36ºC in sistem stationar si sunt urmarite la microscop zilnic pentru aparitia efectului citopatic
- efectul citopatic 4+ (100%) a aparut, atit pe RD, cit si pe L20B la 48 ore de la inoculare, cind placutele au fost puse la -20ºC
5.TITRAREA ANTIGENELOR POLIOVIRUSURILOR SABIN
Titrarea s-a efectuat pe acelasi tip de cultura pe care s-a realizat multiplicarea, prin micrometoda, pe placute cu 96 godeuri .Au fost facute cate 3 determinari pentru fiecare virus, pentru a se obtine o medie a titrurilor cat mai corecta (Tab.4)
Tehnica :se efectueaza dilutii logaritmice in baza 10 ale virusurilor de testat, de la -1 la –8
- se repartizeaza in godeurile placilor cite 25 µl mediu Eagle MEM cu 2% ser fetal
- se inoculeaza dilutiile de la – 3 la – 8, pe cite 16 godeuri fiecare dilutie, 25 µl virus/godeu
- in fiecare godeu se repartizeaza cite 100 µl celule (RD sau L20B)
- placile sunt incubate la termostat la 36ºC timp de 4 zile
- citirea la microscop pentru notarea efectului citopatic se face la 3 si 4 zile
Calculul titrului se face dupa formula Karber :
log CCID50 = L – d (S – 0,5), unde
L = log celei mai mici dilutii utilizate in test
d = diferenta intre log dilutiilor
S = suma proportiilor testelor “positive” (godeuri de prezinta efect citopatic)
|
Serotipul
|
Titrul virusului (log CCDI50 /25µl)
|
|
L20B
|
RD
|
|
Polio 1 Sabin
|
6,43
|
6,25
|
|
Polio 2 Sabin
|
6.02
|
6,37
|
|
Polio 3 Sabin
|
5,74
|
6,30
|
6.VERIFICAREA IDENTITATII LOTURILOR NOI DE POLIOVIRUSURI DE REFERINTA
Verificarea identitatii noilor loturi s-a efectuat prin reactia de seroneutralizare, cu seruri neutralizante specifice de tip antipoliovirus, prin micrometoda, pe placute cu 96 godeuri.Se utilizeaza amestecuri de seruri polio astfel : polio 1+2, polio 1+3, polio 2+3, polio 1+2+3. Serotipul poliovirusului este indicat de godeurile unde nu apare efect citopatic.Tulpinile verificate, atit pe RD, cit si pe L20B s-au dovedit a apartin serotipului corespunzator. Loturile de virus astfel obtinute si verificate au fost repartizate in criotuburi si stocate la -20ºC, urmind a fi utilizate ca martori pozitivi in testele moleculare ce vor fi efectuate.
7.TITRARE TULPINILOR DE POLIOVIRUS IZOLATE IN ANUL 2007
Titrarea tulpinilor de poliovirus izolate in 2007 a fost precedata de multiplicarea acestora pe linia celulara RD prin pasajul acestora de pe linuia L20B .
Tehnica s-au efectuat dilutii logaritmice in baza 10 ale virusurilor de testat, de la -1 la –8
- se repartizeaza in godeurile placilor cite 50 µl mediu Eagle MEM cu 2% ser fetal
- se inoculeaza dilutiile de la – 5 la – 8, pe cite 20 godeuri fiecare dilutie, 50 µl virus/godeu
- in fiecare godeu se repartizeaza cite 100 µl celule (RD )
- placile sunt incubate la termostat la 36ºC timp de 4 zile
- citirea la microscop pentru notarea efectului citopatic se face la 3 si 4 zile
Calculul titrului se face dupa formula Karber :
log CCID50 = L – d (S – 0,5)
Tulpina
|
log CCID50
|
Titrul virusului (log CCDI50
|
|
25/1/07 PV3
|
-5,45
|
10 5,45/ 0,1ml
|
|
52/2/07 PV1
|
-6,75
|
10 6,75/ 0,1ml
|
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GENOTYPING OF POLIOVIRUS STRAINS ISOLATED IN ROMANIA BETWEEN 2007-2010 FROM AFP CASES AND HEALTHY CONTACTS AS A MAIN OBJECTIF OF THE GLOBAL POLIO ERADICATION
Poliomyelitis is an acute paralytic disease caused by three distinct poliovirus serotypes. The goal of the Global Polio Eradication Initiative is to stop the global transmission of poliovirus. The eradication program in most countries needs high routine coverage with at least three doses of oral poliovirus vaccine (OPV). The OPV consists of three live attenuated Sabin poliovirus strains obtained from wild strains by sequential in vitro and in vivo passages . It is safe, induces long-lasting protective systemic humoral and cellular immunity and local mucosal resistance to poliovirus infection, making it the primary tool for poliomyelitis eradication . Extraordinary progress towards eradicating polio has been made. Poliovirus transmission has been stopped in the Americas and the Western Pacific region . Only six countries were still considered endemic by the end of 2003. Europe has been declared “ polio free” since the WHO / EURO meeting on June 5th, 2002.
However, vaccine-associated paralytic poliomyelitis (VAPP) and the development and circulation of pathogenic vaccine-derived poliovirus (VDPV) strains are the major risks of OPV. During their multiplication in humans, Sabin vaccine strains can undergo genetic variation through single or multiple nucleotide substitutions at various sites in the genome or through natural recombination. These are associated with modifications of the original attenuated phenotype, with the OPV strains acquiring wild-strain characteristics, including high pathogenicity . VAPP has been observed in unvaccinated or incompletely immunised children living in close contact with recently vaccinated children and in the vaccinated individuals. Globally, the rate of VAPP is 1 per 750,000 primary vaccinees and 1 per 12 million recipients of two vaccine doses . The described VAPP cases were most frequently associated with serotype 2 and 3 vaccine strains and rarely with the type 1 vaccine strain . In Romania, the risk of recipient VAPP was found to be higher after the introduction of the OPV in 1961 than generally recorded in OPV-using countries. This high rate was found to be associated with multiple intramuscular injections during the incubation period of the OPV strains, and subsequent measures to reduce these simultaneous injections significantly reduced the VAPP rate . Inactivated polio vaccine IPV has recently been included as the first dose of a combined immunisation (IPV followed by OPV) to reduce the number of VAPP cases further. For more than 25 years, the coverage with OPV vaccine in Romania has been greater than 95 %.
The prolonged replication and circulation of VDPVs (OPV strains having more than 1% nucleotide divergence from the original vaccine strains) may also adversely affect the eradication program. Recent outbreaks of poliomyelitis due to circulating VDPVs have been reported in Egypt, Hispaniola, the Philippines and Madagascar These outbreaks occurred in countries with a low vaccine coverage. This may have favoured the multiplication and circulation of OPV strains in unvaccinated children, allowing the genetic and phenotypic drift of vaccine strains towards VDPVs with wild-type poliovirus strain characteristics. The emergence of epidemic and pathogenic VDPVs threaten the final aim of the eradication program, which is the disappearance of poliovirus and the stopping of immunisation. Until now, there is little known about the factors and conditions favouring the emergence and circulation of VDPVs. Therefore, studies to improve surveillance and develop vaccine strategies to limit VDPV spread are urgently needed.
In 2002 in Romania was isolated and characterizated of a type 1 VDPV isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts with low vaccine coverage (~ 50%) from the same socio-cultural group (the Rroma community). Our results show that small populations with a low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. Although this VDPV circulated in at least nine children, only one developed paralytic disease. Host factors, such as age (five months), and genetic factors might have triggered the development of the disease caused by this neurovirulent Sabin 1/Sabin 2/Sabin 1 recombinant strain. All the other children were between one and five years of age, and only one had received two OPV doses .This suggests that surveillance for these strains needs to improve in poliomyelitis-eradicated areas still using OPV. It is often reported that recombinant genomes frequently occur among OPV strains excreted by healthy vaccinated individuals, their contacts in the community, and in patients with VAPP. Genetic recombination appears to be an integral part of poliovirus evolution. Recombination was first observed as part of the natural evolution of polioviruses in vaccine-related isolates with chimeric sequences that were excreted by children exposed to the trivalent OPV. These isolates were detected in healthy OPV recipients and in patients with VAPP. The heterologous sequences of most vaccine-related isolates were derived from the other Sabin OPV strains, with recombinants most frequently being found among serotype 2 and 3 vaccine-related isolates. However, other serotype 1 tripartite Sabin 1/Sabin 2/Sabin 1 intertypic recombinants have been reported, including one found in a case of paralytic poliomyelitis. This suggests that this tripartite arrangement offers a selective advantage to the type 1 virus or that homologous 5’ and 3’ ends are preferred by the type 1 genome and its replication machinery.
Two reported type 1 VDPVs have been shown to be recombinants of Sabin 1 and non-OPV enteroviruses. We do not know yet whether these genetic features or host factors influence the capacity of a strain to multiply better, to persist in the gut, and/or to circulate. Our results confirm that mutant intertypic recombinants that belong to serotype 1 (with the capsid and antigenicity of Sabin 1) and that are products of genetic exchanges with other OPV strains can circulate for a long time in the population.
In our study about the molecular characterization of poliovirus strains by the RT-PCR-RFLP assay and its use in the active surveillance for acute flaccid paralysis cases in Romania between 2001-2006 67 PV strains isolated from AFP, FP cases and healthy contacts were investigated by RFLP reactions. The two regions investigated with RT-PCR-RFLP assay were chosen for the detection of recombinant strains and for confirmation vaccinal origin of strains as for their genotyping. The frequency of appearance and profiles of recombinant genomes in 3D polimerase region for strains investigated was 44% for S3/S2, 17% for S3/S1, 9% for S2/S1, 9% S2/ S1+S2, 4 % for S1/S2, 17% for S1/S1+S3, S3/S1+S2, S3/S2+S3, S2+S3/S1. The frequency of recombinations was 38% for type 1 PV, 40% for type 2 PV and 52 % for type 3 PV. Our study about seroprevalence of antibodies against poliovirus types was showed that the seroprevalence was higher fot type 1 and 2 PV than of type 3. The implication of less seroprevalence o antibodies agains types 3 PV (85,7%) was that most of then poliovirus strains isolated were with type 3 and that was created the local conditions for frequents recombination events between type 3 PV and anothers serotypes. The preferences in the recombination type were S3-like with S2-like, and S3-like with S1-like. One strain VDPV a recombinant Sabin 1/Sabin 2/Sabin1 was isolated in Romania in 2002.
The recombination is involved in the natural evolution of Sabin strains and it was obtained the high frequency of recombinant genome in OPV strains, isolated from AFP cases (45%), FP cases (60%) and healthy contacts (26%). In our study from all VAPP cases only in 2 cases, the RFLP profiles in 3D region were S3/S1, but the profile of the VDPV Sabin1/Sabin2/Sabin1, triple recombinant strain is one proof that is very important the site, the type of recombination, association with the mutation in the capsid coding region and the host factors for a strain to be responsible for disease. In this situation the establishment of sensitive surveillance.
In those parts of the world where the circulation of wild strains of poliovirus has been stopped, the risk of emerging VDPV circulating in routinely OPV-vaccinated populations raises the question of whether immunisation should not be with IPV alone or with a combined IPV/OPV schedule .
This reaffirms the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries and is also a strong argument against continuing to use OPV alone for polio immunisation once circulation of wild polioviruses has stopped.
Methodology of research- Materials and methods
1. Virus isolation and initial characterization.
Samples collected between 2007-2010 (from about 140 AFP cases and from their healthy contacts) will be processed according to a standard protocol for virus isolation and characterization at the NationalReferenceCenter for Enteroviruses in the Cantacuzino Institute, Bucharest, Romania. L20B (a genetically engineered mouse cell line expressing the human poliovirus receptor PVR) and RD cells (derived from a rhabdomyosarcoma) will be inoculate with the samples, as recommended by WHO for human enterovirus (HEV) detection . L20B and RD will grow in monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal calf serum. Once the complete cytopathic effect (CPE) will be obtaine, the infected cells will be harveste and kept frozen (–20°C) until typing.
Identification of viral isolates Viruses isolated on L20B cells and on RD cells will be serotype by neutralization with pools of locally produced poliovirus horse antisera and will be confirme with monotype specific antisera. Our study show that between 2007-2010 can be isolate about 30 poliovirus strains.
2. Molecular characterization
The molecular method will be RT-PCR –RFLP developed by I. Pasteur Paris and we will try to introduce the WHO technique in the National Polio Laboratory. The technique will be apply to 30 poliovirus strains and to Sabin strains. It will be investigated 3 genomic regions for detection point mutations and recombination appeareance.
RT-PCR assay
The viral supernatant diluted 1/10 (2 ml), RNasin 0.5 ml (20 U) and antisense primer 1ml (10 pmol) will be heat at 80°C for 5 min and then annealed by incubation at 50°C for 5 min. A mixture of 4 ml of transcriptase buffer (5X), 1ml of deoxynucleoside triphosphate (10 mM), 1U of avian myeloblastosis virus reverse transcriptase, and distilled water to a final volume of 20 ml will be add to the annealed template-RNA solution. Transcription will be allow to occur by incubation at 50°C for 30 min. The reverse transcription (RT) products will be denaturate at 95°C for 5 min and then immediately placed on ice. The amplified fragment will name according to the corresponding region of the PV genome. PCR amplification will be carried out with 20 ml of the transcription product to which will add 80 ml of the following: 3 ml of Taq buffer 10X with no MgCl2, 1ml (10 pmol) of sense primer, 1.25 U of Taq DNA polymerase, and distilled water to 80 ml. The PCR program have 29 cycles of 95°C for 20 sec, 45°C for 1 min, and 70°C for 1 min, followed by one terminal elongation cycle of 95°C for 20 sec, 45°C for 1 min and 70°C for 10 min. The primers use are:
UG24: (1913)-GACACCATGATTCCCCTTAA-(1932)
UC1: (2881)-GAATTCCATGTCAAATCTAGA-(2862)
UG19: (2870)-GACATGGAATTCACCTTTGTGG-(2891)
UC13: (3648)-TAGTACTTAGCTTCCATGTA-(3629)
UG17: (6536)-TCAGTGGCCATGAGAATGGC-(6555)
UC10: (7464)-TTTTTTTTTTTTTTTTTTTTTTTC-(7441)
RFLP : restriction fragment length polymorphism (RFLP) assaywill be do for three different genomic regions amplified by reverse transcription–polymerase chain reaction (RT-PCR) and 3 different rrestriction enzymes DdeI, HinfI, RsaI.
Nucleotide sequence analysis- only for strains with RFLP modified profils
PCR products will be sequence with the primers used for RT-PCR. Before sequencing, the PCR products will be purifie either directly after amplification on Qiaquick spin columns (Qiagen) or by isolating bands from low-melting-point agarose gels after electrophoresis using standard techniques The 5′-end of the viral genome will be amplificate with the 5′/3′ RACE kit (Roche). PCR products will be sequence using the Big Dye Terminator Cycle Sequencing Ready Reaction kit, following the procedure recommend by Applied Biosystems, Perkin-Elmer. Sequences will be aligne using Clustal W software version 1.6 .
Phylogenetic analysis of nucleotide sequences.
Phylogenetic relationships will be do with the DNADist/Neighbor programs of of PHYLIP (Phylogeny Inference Package) version 3.5
3.Epidemiologycal examination
Recommended WHO Case Definitions
- Suspected case: any case of acute-onset flaccid paralysis (AFP) -including Guillain-Barré syndrome- in a person under 15 years of age for any reason other than severe trauma, or paralytic illness in a person of any age in which polio is suspected. The classification "suspected case" is temporary. It should be reclassified as "probable" or "discarded" within 48 hours of notification.
- Probable case: a case in which AFP is found, and no other cause for the paralysis can be identified immediately. The classification of "probable case" is also temporary; within 10 weeks of onset the case should be reclassified as "confirmed", "compatible", "vaccine-associated" or "discarded."
- Confirmed case: a case with acute paralytic illness with or without residual paralysis, and isolation of wild poliovirus from the stools of either the case or its contacts.
- Polio-compatible case: a case in which one adequate stool specimen was not collected from a probable case within 2 weeks of the onset of paralysis, and there is either an acute paralytic illness with polio-compatible residual paralysis at 60 days, or death takes place within 60 days, or the case is lost to follow-up.
- Vaccine-associated Paralytic Poliomyelitis: a case with acute paralytic illness in which vaccine-like poliovirus is isolated from stool samples, and the virus is believed to be the cause of the disease. There are two possible types of vaccine-associated paralytic poliomyelitis (VAPP): recipient and contact. A case classified as a recipient is a person who has onset of AFP 4 to 40 days after receiving OPV and has neurologic sequelae compatible with polio 60 days after the paralysis began. A case is classified as a contact VAAP when a person who has residual paralysis 60 days after the onset of AFP had contact 4 to 40 days before the paralysis began with a person who received OPV somewhere between 4 and 85 days before the contact’s paralysis began.
- Discarded (Not Poliomyelitis): a case with acute paralytic illness for which one adequate stool specimen was obtained within 2 weeks after onset of paralysis and was negative for poliovirus.
- Vaccine associated paralytic polio (VAPP) is a rare form of poliomyelitis caused by a VDPV. A VAPP case is defined by acute paralytic illness in which vaccine-like poliovirus is isolated from stool samples, and the virus is believed to be the cause of the disease.
Vaccine-derived poliovirus (VDPV) is defined as a live, attenuated strain of the virus contained in the Oral Polio Vaccine which has mutated and reverted to a neurotropic form, capable of causing Vaccine-associated paralytic polio (VAPP). VDPVs differ from the parental Sabin Strains at 1 to 15% of VP1 nucleotides. There are three recognized types of VDPVs:
1. cVDPVs (circulating vaccine-derived polioviruses) that are associated with sustained person-to-person transmission and considered to be circulating in the environment;
2. iVDPVs (immuno-deficient excretors of vaccine-derived poliovirus) isolated from immunodeficient patients who have prolonged infections after exposure to OPV; and a VDPVs which are clinical isolates from patients with no recognized immunodeficiency and not associated with an outbreak, or environmental isolates whose ultimate source has not been identified.
