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J. gen. Virol. (1986), 67, 2093-2102. Printedin Great Britain 2093 Key words: poliovirus/nucleotidesequence~Finland/poliomyelitis The Nucleotide Sequence of a Type 3 Poliovirus Isolated During a Recent Outbreak of Poliomyelitis in Finland By P A M E L A J. H U G H E S , 1 D A V I D M. A. E V A N S , 2 P H I L I P D. M I N O R , 2 G E O F F R E Y C. S C H I L D , 2 J E F F R E Y W. A L M O N D 3 AND G L Y N S T A N W A Y 1. 1Department of Biology, University of Essex, Wivenhoe Park, Colchester C04 3SQ, 2National Institute for Biological Standards and Control, Holly Hill, London NW3 6RB and 3Department of Microbiology, University of Reading, London Road, Reading RG1 5AQ, U.K. (Accepted 12 June 1986) SUMMARY We have cloned and sequenced the complete genome of a strain of poliovirus type 3 (23127) isolated during an outbreak of poliomyelitis in Finland. The genome is 7435 nucleotides long excluding the 3' poly(A) stretch and is 95.5 ~ homologous at the amino acid level to the previously sequenced type 3 strain, P3/Leon/37. The most striking feature of the presented sequence is the extent of amino acid substitutions relative to P3/Leon/37 and other type 3 strains in areas of known antigenic importance. The major antigenic determinant for virus neutralization (site 1), located at residues 89 to 100 of VP1, has three amino acid substitutions and there are six substitutions in site 3, a composite site made up of sequences from VP 1 and VP3. The variation in these regions probably accounts for the observed unusual antigenicity and may explain why the virus was able to spread in a well-vaccinated community. Sequence comparisons imply that the virus is not derived from the currently used live attenuated vaccine. INTRODUCTION Paralytic poliomyelitis has been largely eliminated from developed countries through the use of effective vaccines against the causative agents, the three poliovirus serotypes. However, in late 1984 there was an outbreak of the disease in Finland, a country where inactivated poliovirus vaccines have been used successfully for about 25 years and where the vaccine acceptance rate is in excess of 9 7 ~ (Weekly Epidemiological Record, 1985; Lapinlemu & Stenvik, 1981). Nine paralytic cases occurred, each associated with infection by a poliovirus designated type 3. However, further serological characterization revealed that the viruses isolated during the outbreak were antigenically unusual in their response to both polyclonal and monoclonal antisera raised against the other type 3 strains. This may have contributed to the ability to spread in a well-vaccinated community (Magrath et aL, 1986). The antigenic properties were partly explained by mutations detected in a region of the poliovirus capsid known to play a major role in virus neutralization (Evans et al., 1983). This finding is surprising because wild strains of poliovirus are usually highly conserved in this region (Ferguson et al., 1985). Poliovirus is a member of the picornaviridae family and has a positive-sense, single-stranded RNA genome of approximately 7500 nucleotides, enclosed within a protein capsid made up of 60 copies of each of four proteins, VP1 to VP4. The Y-most 740 nucleotides of the genome are presumed to be non-translated and an AUG located after this region initiates the synthesis of a long polyprotein which is cleaved by virus proteases to generate all the known virus proteins. As part of our research on poliovirus antigenicity and the molecular relationship between picornaviruses, we have determined the complete nucleotide sequence of a strain of the virus responsible for the Finnish outbreak. The sequence presented here shows that the regions of the poliovirus capsid known to be antigenically important exhibit marked differences from the 0000-7209 O 1986 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 2094 P. J. HUGHES AND OTHERS previously sequenced poliovirus type 3 strains and this is consistent with the unusual antigenicity. In addition, the sequence gives an indication of the degree of molecular heterogeneity within a poliovirus serotype. METHODS Virus strain. The virus under study (strain 23127) was isolated during the outbreak from an asymptomatic individual who was a close relative of a victim of paralytic poliomyelitis. Molecular cloning and nucleotide sequencing. Purified viral RNA (5 p-g) was reverse-transcribed and cloned into Escherichia coli JA221 by the cDNA :RNA hybrid method (Cann et al., 1983; Stanway et al., 1984a) using pBR322 as the vector. Of the recombinants obtained, 800 were analysed by hybridization using portions of the previously cloned poliovirus type 3 genome as probes (Cannet al., 1983). A set of five overlapping subgenomic clones were selected which together spanned the genome (data not shown). The cDNA inserts were sequenced by the dideoxynucleotide method after generation of random fragments and cloning into M I3 rap8 as previously described (Stanway et al., 1984b). This methodology generated the majority of the sequence data while residual, small stretches were sequenced after cloning AluI or Sau3AI fragments into M13 mp8 digested with SmaI or BamHI respectively. Approximately 8 0 ~ of the sequence was obtained in both orientations and all regions were sequenced at least twice. Sequence data were assembled and analysed using published computer programs (Staden, 1980). RESULTS AND DISCUSSION The complete nucleotide sequence and predicted amino acid sequence of strain 23127 are shown in Fig. 1. The genome comprises a 5' non-coding region of 746 nucleotides, a single open reading frame of 6618 nucleotides (2206 codons), a short 3' non-coding region of 71 nucleotides and a poly(A) tract. Sequence comparisons indicate that strain 23127 is highly homologous to the representatives of each of the three poliovirus serotypes sequenced to date. These are the type 1 strain P1/Mahoney (Kitamura et al., 1981) and type 3 strain P3/Leon/37 (Stanway et al., 1984b), which are wild-type strains, and the Sabin type 2 vaccine strain P2/P712, Ch, 2ab (Toyoda et al., 1984). The amino acid homologies between the individual proteins of strain 23127 and these viruses are shown in Table 1. Overall, in confirmation of the serotyping, 23127 most closely resembles P3/Leon/37. This is particularly evident in the capsid proteins where the level of homology ranges from 91.7~ in VP1 to 100~o in VP4. The corresponding figures for the VP1 proteins of the type 1 and type 2 strains are only 71.3~ and 73.7~ respectively. Taking the genome as a whole there is 95.5~ amino acid homology and 80.7~ nucleotide homology to P3/Leon/37. Given the immunological relationship between strain 23127 and P3/Leon/37, the degree of nucleotide sequence divergence is quite high. This is consistent with the generally accepted view of the high mutability of an RNA genome (for review, see Domingo et al., 1985). Although strain 23127 is more closely related to P3/Leon/37 than it is to the type 1 and type 2 strains, the pattern of homology is not that which might be expected from previous comparisons between poliovirus strains. In particular, the 5' untranslated region and the non-structural proteins show an unexpectedly high degree of difference to P3/Leon/37. A comparison between the 5' untranslated regions of strain 23127 and the three poliovirus serotypes is presented diagrammatically in Fig. 2 and the degree of homology is shown in Table 2. This region is often highly conserved among related picornaviruses (Stanway et al., 1983, 1984c; Toyoda et al., 1984) and this is also true for strain 23127 and the other polioviruses. The conservation is of the order of 85 ~ in each case. The 100 nucleotides prior to the AUG which initiates the long open reading frame are highly variable between poliovirus serotypes and if this region is ignored the sequence conservation is around 90~. In contrast to the results seen for the capsid proteins, the 5' noncoding region of strain 23127 is not obviously more similar to the type 3 strain in terms of overall homology. Indeed, it is slightly more similar to P 1/Mahoney (Table 2). This is due largely to two particular stretches of nucleotides (positions 122 to 187 and 473 to 508) where strain 23127 is strikingly similar to P1/Mahoney. In the former case there is perfect conservation of 66 nucleotides between these viruses while P3/Leon/37 differs at 13 positions. There is only one difference to P2/P712, Ch, 2ab in the same area. In the stretch of nucleotides from positions 473 to 508 there are only three differences between strain 23127 and P1/Mahoney while there are 14 differences between the type 3 strains. P2/P712, Ch, 2ab differs at 11 positions in this area. Strain 23127 is most similar to P3/Leon/37 outside these regions (Table 2). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 Sequence of a poliovirus Finland strain 2095 Table 1. Percentage amino acid homology between the proteins of strain 23127 and previously sequenced polioviruses VP4 VP2 VP3 VP1 P2-A P2-B P2-C P3-A VPg Protease Polymerase P1/Mahoney 92.7 83.0 82.8 71.3 94.0 84.5 96.0 97.7 95.7 96.7 98.3 P2/P712,Ch, 2ab 92.7 80.0 84.9 73.7 93.3 88.7 96.4 97.7 100 96.2 97.4 P3/Leon/37 100 95.2 95.0 91.7 93.3 91.8 97.3 98-9 95.7 95.6 97.4 Table 2. Percentage nucleotide homology between strain 23127 and other polioviruses in the non- coding regions Overall 5' non-coding Nucleotides 1-640 Excluding nucleotides 122-187 and 473-508 3' non-coding P1/Mahoney 85-9 90.8 89.6 P2/P712,Ch, 2ab 84.0 89.8 91.5 P3/Leon/37 84.7 90.2 93.3 98.6 97.2 98.6 In view of the demonstration that polioviruses can undergo recombination in man (Kew & Nottay, 1985; Minor et al., 1986a), a possible explanation for this pattern of homology is that strain 23127 is a recombinant in which the 5' untranslated region has been derived from a type 1 strain. This is, however, unlikely given that the type 1-specific regions are flanked by regions where strain 23127 is more homologous to P3/Leon/37. Thus, several recombination events would need to have taken place to generate the final virus. An alternative explanation is that the Finnish strain is closer to the poliovirus type 3 progenitor strain than is P3/Leon/37 and that the latter strain has accumulated several additional mutations in these areas since its divergence. The obvious implication of this is that strain 23127 is not on a direct genetic lineage from P3/Leon/37. Since the type 3 vaccine strain used throughout much of the western world was produced from P3/Leon/37 and differs at only 10 nucleotide positions (Stanway et al., 1984b), these results argue that the virus is not vaccine-derived. It is presumably a wild strain which has circulated independently. In contrast to the 5' non-coding region, the 3' non-coding region of strain 23127 sheds little light on the origin of the strain and its relationship to the other polioviruses. This region is highly homologous between the other sequenced polioviruses and this is also observed with strain 23127. There is just one nucleotide difference from the type 1 and type 3 strains and two from the type 2 strain. The open reading frame is terminated by two stop codons in tandem in each of the polioviruses sequenced to date. Interestingly, the single nucleotide substitution in the 3' noncoding region of strain 23127 destroys the second of these stop codons, indicating that this is not required for the viability of the virus. In addition to the 5' non-coding region, the other seemingly anomalous aspect of the presented sequence is the degree of homology in the non-structural proteins. These are highly conserved between the poliovirus type 1 to 3 strains sequenced to date, as might be expected of proteins which perform the same function remote from the immunological pressures upon the capsid proteins. The capsid proteins of strain 23127 are most similar to P3/Leon/37 and presumably in evolutionary terms these two viruses are closely related. It might therefore be expected that their non-structural proteins would be particularly similar. However, this is not the case since strain 23127 shows approximately the same number of amino acid differences from each of the three Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 2096 P. J. H U G H E S A N D O T H E R S TTAAAACA••TCTG•••TTGTT•••ACC•CA•AGG•C•A••TG•C•G•TAGTACACTG•TAT•A•••TA•CCTT•TA•GC•TGTTTTATA 10 20 30 40 50 60 70 80 90 •TCCCTTCCccGTAACTTAGAAGTATACAAGCCAAGTTCAATAGAAGGGGGTACAAACcAGTACCACCACGAAcAAGcACTTCTGTTTcC I00 II0 120 130 140 150 160 170 180 CcGGTGAAGTTGCATAGATTGTTCcCACGGTcGAAAGcGAcTGATCcGTTACCcGCTCATGTACTTcGGGAAGccTAGTATCACTTcGGA 190 200 210 220 230 240 250 260 270 CTCTTCGATGCGTTGCGCTcAGCACTCAAccccAGAGTGTAGCTTAGGTcGATGAGTCTGGACAAAcCTCACTGGCGACAGTGGTcCAGG 280 290 300 310 320 330 340 350 360 cTGCGTTGGcGGCCTACCTGTGGcCcAAAGCcAcAGGACGCTAGTTGTGAACAAGGTGTGAAGAGccTAcTGAGCTAcATGAGAGTCCTC 370 380 390 400 410 420 430 440 450 CGGCCCCTGAATGCGGCTAATcTCAACCAcGGAGCAGGTAGTCAcAAAccAGTGATTTGCCTGTCGTAAcGCGAAAGTCTGTGGCGGAAC 460 470 480 490 500 510 520 530 540 CGAcTACTTTGGGTGTCCGTGTTTcCTTTTATTCTCATTATGGCTGCTTATGGTGACAATCCTAGATTGTTATcATAAAGTGAGCTGGAT 550 560 570 580 590 600 610 620 630 TGGccATCCGGTGAAAATCAAGcAcATTATATATTTGTTTGTTGGGTTTAACcCTcTGATTGcACGcAcTcTTGGCTTGGTTAGGATTAT 640 650 660 670 680 690 700 710 720 ,~VP4 M G A Q V S S Q K V G A H E N S N R A Y G TGTGTGGAAAAGGCACcATAGTTACAATGGGTGcTCAAGTTTCATcCCAAAAAGTTGGAGCCcATGAGAAcTCGAATAGGGcATATGGTG 730 740 750 760 770 780 790 800 810 G S T I N Y T T I N Y Y K D S A S N A A S K Q D Y S Q D P S GTTcTAccATCAATTACAccAcTATCAATTATTAcAAGGATTcAGCAAGcAATGCCGCATCTAAGCAAGAcTATTCCCAGGATcCCTcTA 820 830 840 850 860 870 880 890 900 )VP2 K F T E P L K D V L I K T A P A L N I S P N V E A C G Y S D R AATTCACCGAACCACTGAAGGATGTGTTAATTAAGACAGCAC•AGCTCTCAA•T•TCCAAATGTTGA•G•ATGTGGATACAGCGATAGGG 910 920 930 940 950 960 970 980 990 V L Q L T L G N S T I T T Q E A A N S V V A X G R W P E F I TGTTGCAATTGACCCTAGGCAACTcCACCATAACAACGcAAGAGGcGGCCAATTCAGTGGTcGcGTATGGTcGTTGGCCTGAGTTTATCC i000 i010 1020 1030 1040 1050 1060 1070 1080 RDDEANPVDQPTEPDVAT(~RFYTLDTVMWG GAGATGAcGAAG~AAA~cAG~GGAccAA~cAA~AGAGc~GGA~TAG~AAcTT~TAGGTT~AcAcGcTG~AcAcAGT~ATGTGGGG~A 1090 1100 IIi0 1120 1130 1140 1150 1160 1170 KES@GWWWKLPDALRDMGLFGQNMYYHYLG AAGAGTccAGGGGATGGTGGTGGAAATTAcCAGATGcT•TCAGAGAcATGGGGC•GTTTGGA•AAAATATGTATTAccAcTAcT•AGGGA 1180 1190 1200 1210 1220 1230 1240 1250 1260 R S G Y T V H V Q C N A S K F H Q G @ L G V F A I P E @ C L GATCAGGATATA•AGTGCACGTcCAGTGCAACGCcTCTAAATTTCATCAGGGCTCATTGGGAGTATTCGcCATAccAGAATTCTGTCTGG 1270 1280 1290 1300 1310 1320 1330 1340 1350 A G D S D @ Q R Y T S Y A N A N P G E @ G G K F Y @ Q F N K CTGGGGACAGTGATAcGCAAAGATAcACCAGcTAcGcGAACGCTAACCCAGGTGAGAAAGGAGGGAAGTTTTATGCTCAATTTAATAAAG 1360 1370 1380 1390 1400 1410 1420 1430 1440 D (~) A V T S P K R E F C P V D Y L L G C G V L (~) G N A F V (]~) ATACTGCTGTAACCTCCCCAAAAAGGGAATTTTGTCCAGTAGACTATTTACTAGGATGTGGTGTTTTGATAGGGAATGCTTTTGTGTTCC 1450 1460 1470 1480 1490 1500 1510 1520 1530 P H Q i l N L R T N N S A T ( ~ ) V L P Y V N A L @ I D S M V K CcCACCAGATTATcAACTTAAGAACCAACAACAGTGcCACcCTAGTATTAccCTATGTAAATGCAcTGTcTATAGATTcAATGGTCAAGC 1540 1550 1560 1570 1580 1590 1600 1610 1620 H N N W G I A I L P L S P L D F A Q D S S V E I P I T V T I ACAATAATTGGGGCATTGC~ATTCTCCCCCTGTCACCATTGGATTTTGCACAAGACTCATCAGTGGAGATACCCA~TACAG~CACAATAG 1630 1640 1650 1660 1670 1680 1690 1700 1710 )VP3 APMCSEFNGLRNVTAPKC) QIGLPVLNTPGSN cTCcGATG•GcAG•GAGTTCAA•GGTT•GcG•AAc•TTAcAG•cc•AAA•cTTcAAGGAcTAc•AGT•c•GAA•A•ACcAGGcAG•AAT• 1720 1730 1740 1750 1760 1770 1780 1790 1800 Q Y L T S D N H Q S P C A I P E F D V T P P I D I P G E V K AGTAcCTAACTTcGGACAACCACCAGTCcCCATGTGCAATcCcAGAGTTTGATGTCACGCCACCAATCGACATACCTGGGGAAGTCAAAA 1810 1820 1830 1840 1850 1860 1870 1880 1890 , @ . E 1900 LA~ ~ 1910 o ~. 1920 i P L. 1930 L E ® ~ K R , ~ , 1940 1950 0. 1960 Y 1970 R V® 1980 L S D S A (~) L S (~) P I L C L S L S P A ® D P R L S H T M L G TGAGCGACAGCGCCAACCTGAGCGGTCCAATCTTATGCCTGTCTCTGTCACCGGCAGCAGACCCTCGCTTATCACACACTATGTTAGGGG 1990 2000 2010 2020 2030 2040 2050 2060 2070 E V L ~ Y Y T H W A G S L K F T F L F C G S M M A T G K ( ~ ) L AGGTTTTGAACTACTACAcACACTGGGCTGGATCC•TGAAGTTCACTTTCTTATTCTGTGGCTCTATGATGGCTACTGGAAAGCTGTTA• 2080 2090 2100 2110 2120 2130 2140 2150 2160 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 Sequence ~ a poliovims Fin~nd strata 2097 V A Y A P P G A Q P P T S R K E A M L G T H V l W @ TCGcTTACG••CCTcCGGGAGCA•AACCACCGACCAGTCGCAAGGAGGCAATGCTCGGCACGcATGTCATTTGGGACTTGGGCCTA•AAT 2170 2180 2190 2200 2210 2220 2230 2240 L G L Q 2250 S S C T M V V P W I S N V T Y R Q T T Q D S F T E G CCTCATGTACGATGGTTGTACCGTGGATCAGCAATGTGAcATACAGACAGACTACACAGGACAGTTTTACTGAAGGGGGATACATCAGcA 2260 2270 2280 2290 2300 2310 2320 2330 G Y I S M F Y Q T R I V V P L S T P K @ M @ M L G F V S A C N D F TGTTCTATCAAACCAGAATTGTGGTTCcTTTATCAACCCCAAAGG~GATGGACATGTTGGGGTTTGTTTCAGCGTGcAAcGATTTCAGTG 2350 2360 2370 2380 2390 2400 2410 2420 2430 S 2340 PI V R L L R D T T H I S Q@ A @ P Q ~ @ D L [ @ E V A Q@ A TACGGTTGCTGCGCGA~ACAACACACATTTCGCAGGCGGCCA?GCCACAAGGGG~GGATGATCTGATAA~CGAAGTGGCACAAAATGcTT 2440 2450 2460 2470 2480 2490 2500 2510 2520 L @ L S L P K @ Q @@ L P 0 T K A S G P A H S K E V P @ TAGCTCTCTCACTTcCCAAGCCACAAAgCAATCTTCCTGATACAAAGGCCAGTGGGCC~GCCCACTCCAAGGAAGTTCCGACGCTGACTG 2530 2540 2550 2560 2570 2580 2590 2600 L T 2610 A V E T G A T N P L @ P S D T V Q T R H V @ Q@ R 5 R S E S CGGTAGAGACCGGTGCCACTAATCCACTTGTTCCATCAGACACCGTGCAGACGCGACACGTCATACAGCAGCGTAGCAGGTCTGAGTCAA 2620 2630 2640 2660 2660 2670 2680 2690 2700 T I E S F F A R G A C V A I I E V D N E Q P @ T @ @ CTATTGAATCCTTCTTTGCAAGGGGGGCGTGTGTTGCCATTATTGAGGTGGACAATGAACAGCcAGCCACCAATGTCCAAAAACTGTTCG L F F T I S G D S L Y S A M @ V D D F G V L A @ R V V N D H N P T K V GGGATTcACTcTAcAG~GCcATGG~TGTGGATGATTT~GGTGTCcTCGccATAAGAGTGGTcAATGATcAcAAcc~CACTAAAGTAAcAT 3160 3170 3180 3190 3200 3210 3220 3230 3240 T 2710 2720 2730 274o 275o 2760 2770 Q 2780 A @ W R I T Y K D T V Q L R R K L E F F T Y S R F D cCAcATGGCGAATCACcTATAAGGACACGGTGCAGTTGCGCCGGAAATTAGAGTTCTTCACATACTCCCGCTTTGACATGGAATTTACCT 2800 2810 2820 2830 2840 2850 2860 2870 K 2790 M E 2880 FVVTANFTN@NNGHALNQVYQIMYIPPGAP TTG•GGTGAcAG••AA•TTcAccAA•TCAAA•AA•GGG•ATG•T•TAAA••AGGTCTA•CAGATAATGTAcA••••cc•AGGTG••••TA 2890 2900 2910 2920 2930 2940 2950 2960 T P K S W D D Y T W Q T S S N P S I F Y T Y G A A P CGCCAAAGTCATGGGACGATTAcACCTGGCAAACTTCTTCAAACCCATCCATATTCTAcACCTAcGGTGCTGCCCCGGCCCGGATcTCCG 2980 2990 3000 3010 3020 3030 3040 3050 2970 A R 3060 V P Y V G L A N A Y S H F Y D G F A K V P L K @ D A N D Q @ TACCATACGTGGGATTAGCAAACGCATAcTCGCACTTCTATGACGGTTTTGCTAAAGTACCACTGAAAT~TGATG~AAATGAT~AAGTGG 3070 3080 3090 3100 3110 3120 3130 3140 3150 SKVR@YMKPKHVRVWCPRPPRAVPYYGPGV cAAAGGTGCGGG•GTACATGAAA•CCAAG•ATG•GAGAGTGTGGTG•cAAGAC•c•GCGGG•GG•A•GTA•TACGGGCCAGGAGTGG 3250 3260 3270 3280 3290 3300 3310 3320 )P2-A D Y K @ @ L @ P L S E K G L T T Y I G F G H Q N K A V ACTACAAAGACGGGTTGGCAccTCTATCAGAGAAGGGACTCACAACGTATGGGTTTGGTCACCAAAATAAAGCTGTTTAcACAGCGGGTT 3330 A G Y K I C N Y H L A T ~ E D L Q N @ V S @ M W N R D L L V @ ATAAGATcTGCAATTACCACCTCGCTACAC~AGAAGATCTACAGAATG~AGTGAGCGTCATGTGGAATAGGGATCTCCTGGTGACAGAAT 3430 3440 3450 3460 3470 3480 3490 3500 3510 E S K A Q G @ D S I A R C N C @ @ G V Y Y C E S R @ @ Y Y P CAAAAGCTcAGGGCATAGATTcTATcGccAGATGCAATTGCA~CACTGGCGTGTATTATTGTGAGTCCAGAA~TAGGTATTACCCTGTTT 3520 3530 3540 3550 3560 3570 3580 3590 3600 V 3340 3350 3360 3370 3380 3390 3400 Y 3410 S F V G P T F Q Y M E A N D Y Y P A R Y Q S H M L I CCTTCGTTGGTCCAACTTTCCAATACATGGAAGCAAATGATTACTA••CAGCTCGATACCAGTcCCACATGCTAATCGGTCACGGTTTCG 3610 3620 3630 3640 3650 3660 3670 3680 T 3420 G H G F F G 3690 ASPGDCGGILRCQHGVIGI@TAGGEGLVAF CA••A••TGGGGATTGTGG•GGTAT••TCAGATG••AG•ATGG•GTGATAGGCATCATCACGGCTGGAGGAGAGGGC•TGGTG•CTTTCT 3700 3710 3720 3730 3740 3750 3760 3770 3780 [--.>P7-8 S D I R D L Y A Y E E E A M E Q I G I S @ Y @ E S L G CAGATATTAGAGACCTTTATGCATATGAGGAAGAAGCCATGGAGCAGGGCATTTCCAGCTACGTTGAGTCC•TTGGAGCAGCATTTGGAA 3790 3800 3810 3820 3830 3840 3850 3860 A A S G F T Q Q I G D K I @ E L T @ M V T S T I T E K L GCGGGTTCACCCAGCAAATCGGGGATAAAATTATTGAGCTAACCGGCATGGTCACCAGCACTATAACCGAAAAACTATTGAAGAACTTAA 3880 3890 3900 3910 3920 3930 3940 3950 L 3870 K N L 3960 I K I Q S S L V I I T R N Y Q D T T T V L A T L A L L G C D TAAAGATTGTAT•CTCcCTAGTCATTATCACTAGGAATTATGATGACACCACCACTGTTTTAGCAACCCTAGCGcTAcTTGGGTGTGATG 3970 3980 3990 4000 4010 4020 4030 4040 4050 )P2-C V S P W Q W L K K K A C D ~ L E I P Y V Q R Q I G D s W L K K TGTCTCCTTGGCAGTGGcTCAAAAAGAAAGCTTGTGATA~ACTAGAAATCCCATACGTCATGCGCCAGGGTGACAGCTGGTTGAAGAAGT 4060 4070 4080 4090 4100 4110 4120 4130 4140 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 2098 P.J. HUGHES AND OTHERS F T E A C N A A K G L E W V S N K I S K F I D W L R TTACGGAAGcATGCAAcGCTGcCAAAGGACTGGAATGGGTGTCCAACAA•ATCT•CAAATTTATT•ATTGGTTGAGAGAGAAAATCATTC 4!50 4160 4170 4180 4190 4200 4210 4220 E @ I I L V 4230 PQARDKLEFVTKLKQLEMLENQi~TIHQSC •••AAG•TAG•GA•AAATT•GAA•TTGTTAccAAGTTGAAA•AGTTAGAGATGTTGGAGAAC•AAATT••AAcCAT•CA••AGT•ATGCC 4240 4250 4260 4270 4280 4290 4300 4310 4320 P S Q E H Q E I L F N N V R W L S I Q S K R F A P L Y A L E CCAGCCAGGAAcAcCAGGAGATTcTGTTCAAcAATGTGAGGTGGTTGTCCATTCAGTCAAAAAGGTT•GCCCCACTCTACGCATTAGAGG 4330 4340 4350 4360 4370 4380 4390 4400 4410 A K R I Q K L E H T I N N Y I Q F K S K H R I E P V CAAAGAGAATACAGAAGTTGGAACACACGATTAACAATTACATACAGTTCAAGAGCAAACACCGTATTGAACCAGTATGTTTGTTA•TAC 4420 4430 4440 4450 4460 4470 4480 4490 C L 4500 ~ G S ~ G T G K S ' V A ~ R L I A R A I A E K ~ N I S I ~ S L ATGGTAG•CCCGGAACTGGcAAGTCGGTAGCCACcAACCTAATAGCTAGGGCTATAG•GGAGAAGGAAAACAccTCCACATACTCATTAC 4510 4520 4530 4540 4550 4560 4570 4580 4590 P P D P S H F D G Y K Q Q G V V l M D D L N Q N P D G A D M cACCAGATCcATCTCATTTCGACGGGTACAAGCAACAGGGAGTAGTGATCATGGATGATCTAAACCAGAATCcAGATGGAGCCGATATGA 4600 4610 4620 4630 4640 4650 4660 4670 4680 K L F C Q M V S T V E F I P P M A S L E E K G I L F T S N Y AATTATTCTGTcAAATGGTTTCCACAGTGGAGTTCATAccAccAATGGCTTCACTTGAAGAGAAAGGCATTCTATTTACATCAAATTACG 4690 4700 4710 4720 4730 4740 4750 4760 4770 V L A S T N S S R I T P P T V A H S D A L A R R F A F D M D TCCTAGCCTCTACCAACTCTAGCCGTATCACGCCACCCACTGTTGCACACAGTGACGCTTTGGCcAGGCGTTTTGCATTcGAcATGGACA 4780 4790 4800 4810 4820 4830 4840 4850 4860 I Q V M Q E Y S R D G K L N M Q M A T E ~ C K @ C H TTCAAGTcATGAGTGAGTACTCCAGGGATGGGAAATTAAACATGACAATGGCAACAGAAATGTGCAAGAACTGTCATCAACCAGCAAACT 4870 4880 4890 4900 4910 4920 4930 4940 Q P A N 4950 FKRCCPLVCGKAIQLMDKSSRVRYS@DQIT ••AAGAGG•G••G•ccA••GG•ATGTGGGAAGGcTATT•AATTAA•GGAcAAG•ccT••AGGGTcAGGTA•AGcATIGAT•AGATcAc•A 4960 4970 4980 4990 5000 5010 5020 5030 5040 i---.--~P 3-A T M I @ N E @ N R R S N I G N C M E A L F Q I G P L Q Y K D L cCATGATAGTTAATGAGA~AAAcAGAAGGTCCAACATTGGTAACTGCATGGAAGcACTGTTTCAAGGCCCTTTACAATACAAGGATTTAA 5050 5060 5070 5080 5090 5100 5110 5120 5130 KIDIKT@PPPECINDLLQAVDSQEVRDYCE AAATTGACATCAAGA~A~TCC~C~GC~CGAGTG~ATAAACGACTTG~TCCAGG~CGTTGACTCT~AAGAAGTGAGGGACTACTGTGAGA 5140 5150 5160 5170 5180 5190 5200 5210 K K G W I V N I T S Q V Q T E R N I N R A M T I L Q AAAAGGGATGGATTGTCAACATCACCAGTCAAGTTCAAACTGAGAGGAACATAAACAGAGCAATGACTATCTTGCAAGCCGTCACCACAT 5230 5240 5250 5260 5270 5280 5290 5300 )VPg 5220 A V T T K R 5310 FAAVAGVVYVMYKLFAGHQIGAYTGLPNKRP TTGCAGCAGTTG~TGGGGTGGTGTATGTGATGTAcAAGCT~TTTGcA~AcAccAAGGGGCTTATACAGG~CTTC~AAA~AGAGA~C~A 5320 5330 5340 5350 5360 5370 5380 5390 5400 >PROTEASE N V P T I R @ A K V Q I G P G F D Y A V A M A K R N I @ T A T ACGTCCCTAcCATTAGAACAGCTAAGGTTCAGGGTCCAGGGTTTGATTATGcAGTGGcCATGGCCAAGAGAAATATTCTTACAGCAAccA 5410 5420 5430 5440 5450 5460 5470 5480 5490 TSKGEFTMLGVHDNVAILPTHASPGE@IVI •cAGTAAAGGAGAA•TcA•GATG••TGGGGTG•AcGA•AAcGTAG•TATT•TA••AA•C•AT•cATCG••AGGTGAGACGATAGTCATTG 5500 5510 5520 5530 6540 5550 5560 5570 5580 D G K E @ E @ L D A K A L E D Q A G T N L E I T I @ ATGGCAAGGAAATT•AAGTGTTAGATGCTAAAGCTCTAGAAGATCA•GCCGGAACTAACTTGGAAATTACTATTGTTACTCTTAA•AGGA 5590 5600 5610 5620 5630 5640 5650 5660 T N E K F R D I R @ H I P @ Q I T E T N D G V L I V ATGAAAAATTTAGAGACAT•AGG•CACACATTCCCGCACAAAT•ACCGAGACAAATGATGGAGTGTTGAT•GTGAACACTAGTAAGTACC 5680 5690 5700 5710 5720 5730 5740 5750 T N L 5670 S K Y P I 5760 PNMYVPVGAVTEQGYLNLGGRQTAR@LMYN c•AA•A•GTATGT•c•TG•AGGTGCTGTGAc••AA•AGGGATA•cTTAAT•TcGGTGGAcGTcAAA•TGcTcGTACA•TAATGTA•AACT 5770 5780 5790 5800 5810 5820 5830 5840 5850 F P T R A G Q C G G V I T C T G K V I G M H V G G N G S H G TCCcAACCAGAGcAGGCCAGTGTGGTGGAGTCATCACATGCACTGGTAAGGTCATTGGGATGCATGTTGGTGGGAACGGTTcACACGGGT 5860 5870 5880 5890 5900 5910 5920 5930 5940 >POLYMERASE F A A A L K R S Y F T Q S Q I G E ] Q W M R P S K E ~ G Y TTGCAGCAGCCTTAAAACGGTCATACTTCACT•AAAGTCAAGGAGAAATCCAATGGATGAGACCATCAAAAGAGGTGGGCTACCCTATAA 5950 5960 5970 5980 5990 60~0 6010 6020 6030 INAP@KTKLEPSAFHYVFEGVKEPAVLTKN ~TAACGCCC~AT~AAGA~AAATTGGAACC~AGTGCATTCCA~TACGTATTTGAAGGTGTTAAGGAAC~AGC~GTG~TCACAAAAAATG 6040 6050 6060 6070 6080 6090 6100 6110 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 6120 Sequence of a poliovirus Finland strain 2099 D P R (~) K T (~) F E E A ! F S K Y V G N K I T E V D E Y M K E ACCCAAGATTCAAAACGGGTTTTGAGGAAGCCATTTTTTCTAAATATG TGGGCAACAAGATTACAGAAGTGGAIGAATATATGAAGGAAG 6130 6140 6150 6160 6170 6180 6190 6200 6210 A V D H Y A G Q L M S L D I (~) T E Q M C L E D A M Y G T D G CAGTGGATCATTATGCTGGCCAGCTCATGTCATTGGATATTAACACTGAGCAAATGTGCCTTGAGGATGCCATGTACGGTACTGATGGTT 6220 6230 6240 6250 6260 6270 6280 6290 6300 L E A k D L S T S A G Y P Y V (7) M G K K K R D I L N K Q T R TGGAAGCACTAGATCTCAGCACAAGCGCGGGGTACCCATATGTTACCATGGGAAAGAAAAAGAG AGACATCCTAAACAAACAAACTAGAG 6310 6320 6330 6340 6350 6360 6370 6380 6390 v D T K E M Q R k L D (~) Y G I N L P L V T Y V K D E L R S K T ATACAAAAGAGATGCAGAGACTTC TGGACACATACGGAATTAAC CTTCCGCTAGTCACTTATGTGAAAGATGAACTAAGATCTAAGACCA 6400 6410 6420 6430 6440 6450 6460 6470 6480 K V E Q G K S R L I E A S S L N D S V A M R M A F G N L Y A AAGTGGAACAGGGAAAATCCAGGCTGATTGAAGCATCCAGTTTAAATGAC TCTGTGGCAATGAGAATGGCGTTTGGTAACTTATACGCTG 6490 6500 6510 6520 6530 6540 6550 6560 6570 A F H (~) N P G V V T G S A V G C D P D L F W S K I P V L M E CTTTCCACAAAAACCCAGGAGTTGTAACAGGCTcAGCAGTTGGATGTGATCCAGATTTGTTTTGGAGTAAAATACCCGTCTTAATGGAAG 6580 6590 6600 6610 6620 6630 6640 6650 6660 E K L F A F D Y T G Y D A S L S P A W F E A L K M V k E K I AAAAACTCTTTGCATTTGACTACAC AGGGTACGACGCCTCGCTCAGCCCGGCCTGGTTTGAAGCACTAAAAATGGTGTTGGAAAAGATAG 6670 6680 6690 6700 6710 6720 6730 6740 6750 G F G 0 R V D Y I D Y L N H S H H L Y K N K (~) Y C V K G G M GCTTTGGAGACAGGGTGGACTACATTGACTAC CTGAACCATTCACATCATCTTTACAAAAAC AAAACATATTGTGTCAAGGGTGGCATGC 6760 6770 6780 6790 6800 6810 6820 6830 6840 P S G C S G T S I F N S M I N N L I I R T L L L K T Y K G I CATCTGGCTGCTCTGGCACCTCTATTTTCAACTCAATGATTAACAACTTGATAATCAGGACACTTTTACTAAAAACCTAC AAGGGCATAG 6850 6860 6870 6880 6890 6900 6910 6920 6930 D L D H L K M I A Y G D D V I A S Y P H E V D A S L L A Q S AC TTAGATCATCTAAAGATGATTGCCTATGGTGATGATGTAATTGCATCCTACCCCCATGAGGTTGACGCTAGTCTCCTAGCCCAATCAG 6940 6950 6960 6970 6980 6990 7000 7010 7020 G K D Y G L T M T P A 0 K S A T F E T V T W E N V T F L K R GAAAAGAC TATGGATTAACCATGACACCAGCTGATAAGTCAGCCACATTTGAAACTGTCACATGGGAGAATGTAACCTTTTTGAAAAGAT 7030 7040 7050 7060 7070 7080 7090 7100 7110 F F R A 0 E (~) Y P F L I H E S I R W T K D H P V M P M K E I TCTTCAGAGCAGATGAGAGGTACCCATTCTTGATCCACCCAGTAATGC CAATGAAGGAAATTCATGAATCAATTAGATGGACCAAAGATC 7120 7130 7140 7160 7160 7170 7180 7190 7200 P R N r Q D H V R S L C L L A W H N G E@E Y N K F L A(~)I CCAGAAACACTCAGGATCACGTCCGCTCTCTGTGCCTATTGGCTTGGCAC AATGGAGAGGACGAATACAATAAATTTTTAGCTATGATCA 7210 7220 7230 7240 7250 7260 7270 7280 7290 R S V P I G R A L L L P E Y S T L Y R R W L D S F * GAAGCGTGCCAATTGGAAGGGCTCTATTGcTCCCTGAGTACTCTACATTGTACCGCCGTTGGCT•GAcTCTTTTTAGCAACCCTAC•TCA 7300 7310 7320 7330 7340 7350 7360 7370 7380 GTCGAATTGGATTGGGTCATACTGTTGTAGGGGTAAATTTTTCTTTAATTCGGAG-poly 7390 7400 7410 7420 7430 A Fig. I. Complete nucleotide sequence and predicted amino acid sequence of strain 23127, a type 3 poliovirus isolated during the recent outbreak of poliomyelitis in Finland. Amino acid differences from the previously sequenced type 3 strain, P3/Leon/37, are circled. poliovirus serotypes in this region (Table 1). Furthermore, differences in the non-structural region account for virtually half the coding differences between the two type 3 strains. There are at least two explanations for these surprising observations. Again it could be thought that strain 23127 is a recombinant, this time between a type 3 strain and an unknown enterovirus which has donated the region corresponding to the non-structural proteins. Alternatively there may be a number of positions in the sequences of the non-structural proteins which are highly tolerant of amino acid substitutions, and with the high mutability of the R N A genome differences quickly accumulated even between the otherwise closely related P3/Leon/37 and strain 23127. Partial sequencing of further field strains of poliovirus type 3 should give a clearer picture of the relationship between strains within the same poliovirus serotype. From the point of view of the evolutionary relationship between strain 23127 and P3/Leon/37, it is interesting to note that of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 2100 P. J. HUGHES AND OTHERS llllllllllllHI 1ill1 1ill I 11111111111111 I Ilfl Illl I Illllll illllIIIl11111IIIIII I IIIIIIIIIIIIIIIIIIIIIIIIIII II III IIIIi flIT llllIl llll IIII 1111III lllUUl IHlllllIl lllJl IIIIIIIIIII llllllllJI lil~l IIIllillll llllI IIIIII IIIIIIIIIII IIIII II UllIIIIIIIIIIIIII IIIIII lllll IIII IIII U IIIII IIII 11'I' IIIII I I I I I I I I 100 200 300 400 500 600 700 Fig. 2. Comparison between strain 23127 and previously sequenced poliovirus strains in the 5' noncoding region. Vertical bars represent nucleotide differences. The strains used for the comparison are: 1, P1/Mahoney; 2, P2/P712, Ch, 2ab; 3, P3/Leon/37. Nucleotide numbers (Fig. 1) are indicated. the 49 amino acid differences in the non-structural region, at 17 positions the Finnish strain has the same sequence as both the type 1 and the type 2 strains. This seems to imply that at these positions mutations have occurred in the P3/Leon/37 lineage since its divergence from the lineage which eventually produced strain 23127. This again suggests that strain 23127 is not descended from P3/Leon/37 or from the vaccine strain P3/Leon 12alb. In the capsid proteins, the relationship between strain 23127 and the previously sequenced strains of poliovirus is clearer than in other areas of the genome. Here, there are 50 amino acid differences from P3/Leon/37, which is only about one-third of the differences from P 1/Mahoney and P2/P712, Ch, 2ab and the amino acid homologies are much greater within each of the individual proteins (Table 1). In addition, there is a very clear relationship to P3/Leon/37 in regions where there is hypervariability between the three previously sequenced strains such as the antigenic sites and the N terminus of VP 1. This accounts for the serotyping of 23127 and the other Finnish strains as type 3 polioviruses. Variations between the type 3 strains seem to be scattered throughout the capsid proteins, with the exception of VP4 where there are no amino acid differences. This may be due to the fact that VP4 is apparently not exposed on the surface of the virus particle and is therefore not subject to immunological pressure (Hogle et al., 1985). When the differences were plotted on the recently derived three-dimensional structure of P1/Mahoney (Hogle et al., 1985), the great majority were found to be in loops and other elaborations outside the fl-barrel core structure of the three major capsid proteins, VP 1 to VP3 (data not shown). This is consistent with the #-barrel structure being of central importance in determining the morphology of the capsid. Another feature of the sequence comparison with P3/Leon/37 is the concentration of amino acid differences in the N-terminal region of VP 1. This part of the protein lies within the interior of the virus particle and therefore cannot be involved in the antigenicity of the virus. However, it is highly variable between the known polioviruses and is possibly flexible in terms of conformation since its structure is not observable by X-ray crystallography (Hogle et al., 1985). This possibly explains why a number of amino acid differences have been able to accumulate between the two type 3 strains. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 Sequence of a poliovirus Finland strain 2101 Table 3. Amino acid sequences of P3/Leon/37 and strain 23127 in regions of antigenic importance Sequence* P3/Leon/37 (EVDNEQP_TTRAQ) 23127 (EVDNEQPATNVQ) Site 2a VP1 : 217-223 P3/Leon/37 TDA(NDQ)_I 23127 SDA(NDQ)V Site 2b VP2:164--172 P3/Leon/37 (_~AVTSPKRE) 23127 (.T_AVTSPKRE) Site 3a VPI: 286-290 P3/Leon/37 (KNNLD_) 23127 (KDGL_A) Site 3b VP3: 58-60, 70, 71, 77, 79, 80 P3/Leon/37 (EST-VT-D-S)O 23127 (ENT-VR-N-S)G * The previously identified antigenic sites (Minor et al., 1986b) are enclosed in parentheses. Site 1 Location VPI: 89-100 Extensive analysis of the antigenicity of polioviruses in the past has revealed the presence of several regions important in eliciting neutralizing antibodies (Evans et al., 1983; Minor et al., 1986b). The Finnish strain shows amino acid sequence differences from P3/Leon/37 in many of these regions (Table 3). As already observed (Magrath et al., 1986), the major antigenic site for poliovirus neutralization, termed site 1 and located at positions 89 to 100 in VP1, contains three amino acid substitutions in strain 23127. Our results show that there are also drastic differences in a composite antigenic site (site 3; Minor et al., 1986b), made up of amino acids 286 to 290 of VP1 and several amino acids from VP3 (positions 58 to 60, 70, 71, 77, 79). Strain 23127 shows six amino acid substitutions relative to P3/Leon/37 in this antigenic site. These are at positions 286, 287 and 289 of VP1 and positions 59, 71 and 77 of VP3. In addition, position 80 of VP3 is different. While this residue has not previously been included in the antigenic site, its close proximity suggests that a mutation here may also affect antigenicity. Overall, more than half the residues known to be involved in this antigenic site are different from those of P3/Leon/37. It would therefore be expected that this site would not be recognized by antibodies directed against the corresponding site in P3/Leon/37. In type 3 poliovirus, this antigenic region is far less immunodominant than site 1 (Minor et al., 1986b) but these differences must contribute, to some extent, to the distinctive overall antigenicity of strain 23127. Interestingly, two of the different amino acids (positions 286 and 287 of VP1) are identical to the corresponding amino acids of P1/Mahoney and P2/P712, Ch, 2ab. It is possible that this is a similar situation to that of the homology in the non-coding region described above which suggested that strain 23127 is closer to a type 3 progenitor than is P3/Leon/37. The observation adds further weight to the idea that strain 23127 is not on a direct lineage from P3/Leon/37 and that it is not vaccine-derived. The homologous two amino acids are followed by an amino acid insertion in P 1/Mahoney relative to both of the type 3 strains and to P2/P712, Ch, 2ab. The insertion event presumably occurred after the divergence of the poliovirus serotypes. The other antigenic site identified on the poliovirus capsid, site 2, is also composite and is made up of amino acids 220 to 222 of VP1 and, in poliovirus type 3, residues 164 to 172 of VP2 (Minor et al., 1986 b). Surprisingly, these regions are highly conserved in strain 23127, the single amino acid difference from P3/Leon/37 being located at position 164 of VP2. Although the VP1 component of this site is identical in the two type 3 strains, two changes are seen flanking the region (at positions 217 and 223) and these may affect the antigenicity. However, substitutions in this antigenic site would seem to play less of a role in the overall antigenic differences than substitutions in site 1 and site 3. Taken together, these amino acid substitutions in the three areas of known antigenic importance are consistent with the unusual antigenicity of the virus and probably contributed to its ability to spread in a well-vaccinated community. Finally, there is some suggestion that the virus drifted antigenically during the outbreak since some of the viruses isolated show only one or two amino acid differences to P3/Leon/37 in the region of the major antigenic site, rather than the three seen in strain 23127 (Magrath et al., 1986). It would be interesting to analyse antigenic sites 2 and 3 of these isolates, to monitor the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19 2102 P.J. HUGHES AND OTHERS extent o f drift there. I f such drift has o c c u r r e d o v e r the relatively short p e r i o d o f the o u t b r e a k it w o u l d i n d i c a t e t h a t the poliovirus c a p s i d is able to a c c o m m o d a t e relatively readily the a m i n o acid differences required. It is thus surprising t h a t to d a t e only three poliovirus serotypes h a v e been identified and t h a t wild strains are usually c o n s e r v e d antigenically. It r e m a i n s to be seen w h e t h e r the circulating viruses will c o n t i n u e to d i v e r g e and w h e t h e r ultimately a n e w poliovirus serotype will emerge. T h i s w o u l d h a v e i m p o r t a n t i m p l i c a t i o n s for the future r e q u i r e m e n t s for poliovirus vaccines. This work was supported by the Medical Research Council, grant number G83242556CB. REFERENCES CANN, A. J., STANVCAY,G., HAUPTMANN, R., MINOR, P. D., SCHILD, G. C., CLARKE, L. D., MOUNTFORD, R. C. & ALMOND, J. w, (1983). 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C., MINOR, P. D., HORAUD, F., CRAINIC, R., STENVIK, M. & HOVl, T. (1986). Antigenic and molecular properties of type 3 poliovirus responsible for an outbreak of poliomyelitis in a vaccinated population. Journal of General Virology 67, 899-905. MINOR, P. D., FERGUSON, M., EVANS, D. M. A., ALMOND, J. W. & ICENOGLE, J. P. (1986b). Antigenic structure of polioviruses of serotypes 1, 2 and 3. Journal of General Virology 67, 1283-1291. STADEN,R. (1980). A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Research 8, 3673-3694. STANWAY, G., CANN, A. J., HAUPTMANN, R., HUGHES, P., CLARKE, L. D., MOUNTFORD, R. C., MINOR, P. D., SCHILD, G. C. &ALMOND,J. W. (1983). The nucleotide sequence of poliovirus type 3 Leon 12atb: comparison with poliovirus type 1. Nucleic Acids Research ll, 5629-5643. STANWAY, G., MOUNTFORD, R. C., COX, S. D. J., SCHILD, G. C., MINOR, P. D. & ALMOND, J. W. (1984a). 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WEEKLYEPIDEMIOLOGICALRECORD(1985). 2, 11. (Received 18 April 1986) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 13:16:19