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Volume 14, Number 11—November 2008
Letter

Paralysis Case and Contact Spread of Recombinant Vaccine–derived Poliovirus, Spain

Ana Avellón1, Maria Cabrerizo1, Teresa de Miguel, Pilar Pérez-Breña, Antonio Tenorio, Jose Luis Pérez, Maria Victoria Martínez de Aragón, and Gloria Trallero
Author affiliations: 1These authors contributed equally to this article.; Instituto de Salud Carlos III National Centre of Microbiology, Madrid, Spain (A. Avellón, M. Cabrerizo, T. de Miguel, P. Pérez-Breña, A. Tenorio, G. Trallero); Instituto de Salud Carlos III National Centre of Epidemiology, Madrid (M.V. Martínez de Aragón); Son Dureta Hospital, Palma de Mallorca, Spain (J.L. Pérez);

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Table

Laboratory methods used for study of vaccine-derived poliovirus case, Spain, 2005*

procedure Test Method† Sample
Sample preparation
 Concentration of sewage for detection of enterovirus in the environment Concentration with negative charge filters (Millipore, Billerica, MA, USA; 0.45 μm) of 20 L of local sewage E
RNA purification from samples (before molecular analysis)
 MagAttract Virus Mini-Biorobot (QIAGEN GmbH, Hilden, Germany) from 200 μL of stool sample dissolved in water
 S
Classic virology techniques
 Cellular culture (Biosafety Level 3) for growing PV LB20 (transgenic mouse), RD (human rhabdomyosarcoma), HEF (human fibroblast), A-549 (ATCC-CCL185) S, E
Immunofluorescence of infected cells Lim-Beyesh-Melnick A-H and RIVM A-G pools I
EV neutralization assay
 Antibodies (Chemicon, Temecula, CA, USA)
 I
Molecular techniques Molecular EV detection RT-nested PCR 5′ UTR (4) S, I, E
Molecular EV quantification MutaReal EV real-time PCR kit (Immunodiagnostik AG, Bensheim, Germany) S
Molecular EV typing RT-nested PCR in major VP1 region (5) S, I, E
PV intratypic characterization Specific vaccine PV RT-PCR (6) S, I, E
PV genome sequencing fragment 1 1s: TAAAACAGCTCTGGGGTTGTA (2–22)
1as: CACCACCCAAGAAGCGGCC (1023–1041)
1ns: GCTCTGGGGTTGTACCCACTCC (9–30)
1nas:TAACTCTGGGCAATTCAACGA(1001–1021) S, I
PV genome sequencing fragment 2† 2s: CATGCTAAACTCCCCAAAC (945–963)
2as: AGGTGCGCAACATGATGG (1882–1910) S, I
PV genome sequencing fragment 3† 3s: CAGACAATTACCAGTCTCC (1814–1832)
3as: ATTACTAAAAATGCATTGGTTCCC (2518–2541) S, I
PV genome sequencing VP1 fragment† VP1s: ACAACACACATTAGTCAAGAGGCTA (2449–2473)
VP1as: GGATTTGGACACCAAAACAAAGC (3385–3407) S, I
PV genome sequencing fragment 4† 4s:GTGCCCACGACCTCCA (3288–3303)
4as: CTTGGGTGCGACATCTCA (4042–4059) S, I
PV genome sequencing fragment 5† 5s: TAATCAAAATTATCTCATCACTTGTG (3962–3987)
5as: CATGAGCGAGTACTCCAGA (4872–4889) S, I
PV genome sequencing fragment 6† 6s: CTGGCCAGGAGATTCG (4834–4949)
6as: AAATGATGGAGTTTTGATCGT(5725–5747) S, I
PV genome sequencing fragment 7† 7s: AGGCAGGAACTAATCTTGAAA (5630–5650)
7as: CTAAGTATGTAGGCAACAAGAT (6164–6185) S, I
PV genome sequencing fragment 8† 8s: CAAAAATGATCCCAGGCTCA (6117–6136)
8as: AAACCTACAAGGGCATAGATT (6917–6937) S, I
PV genome sequencing fragment 9† 9s: CAGGCACATCAATTTTTAACTC (6857–6878)
9as: GGTAAATTTTTCTTTAATTCGGGG(7416–7439) S, I
Additional PV sequencing primers 447as: CCGGCCCCTGAATGCGGC (447–464)
4666s: CCAGACGGAGCAGACATG (4666–4683) S, I

*E, local sewage; S, stools; I, isolates; EV, enterovirus; PV, poliovirus; UTR, untranslated region; VP1, virus capsid protein.
†Sense (s) and antisense (as) primers: 5′ → 3' sequence (position according to X00595). n, nested. All reverse transcription–PCR (RT-PCR) systems had the same conditions: 5 μL of clinical samples (case) or isolates (contacts) were added to the reaction mixture (final volume 50 μL): AMV/Tfl 1X reaction buffer, 2 mmol/L MgSO4, 200 μM each dNTP, 1 μM each primer, 5 U of AMV RT, and 5 U of Tfl DNA polymerase (Access RT-PCR System, Promega, Madison, WI, USA). First RT step of 45 min at 48°C, 2 min at 94°C, 45 cycles of denaturation (94°C, 2 min), annealing (53°C, 1 min), and elongation (68°C,1 min 30 s).

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1These authors contributed equally to this article.

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