Download The Nucleotide Sequence of a Type 3 Poliovirus Isolated During a

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
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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).
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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
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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
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900
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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
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1930
L
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1940
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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
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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
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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
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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 @
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3430
3440
3450
3460
3470
3480
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3500
3510
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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
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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
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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
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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.
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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
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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
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J. w, (1983). Poliovirus type 3: molecular cloning of the genome and nucleotide sequence of the region
encoding the protease and polymerase proteins. Nucleic Acids Research 11, 1267-1281.
DOMINGO, E., MARTINEZ-SALAS, E., SOBRINO, F., DE LA TORRE, J. C., PORTELA, A., ORTIN, I., LOPEZ-GALINDEZ, C.,
PEREZ-BRENA, P., VILLANUEVA, N., NAJERA, R., VANDEPOL, S., STEINHAUER, D., DEPOLO, N. & HOLLAND, J. (1985).
The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance a review. Gene 40, 1-8.
EVANS, D. M. A., MINOR, P. D., SCHILD, G. C. & ALMOND, J. W. (1983). C r i t i c a l role o f a n e i g h t a m i n o a c i d s e q u e n c e o f
VP1 in neutralization of poliovirus type 3. Nature, London 304, 459-462.
FERGUSON, M., EVANS, D. M. A., MAGRATH, D. I., MINOR, P. D., ALMOND, J. W. & SCHILD, G. C. (1985). Induction of
broadly reactive, type specific neutralizing antibody to poliovirus type 3 by synthetic peptides. Virology 143,
505-515.
HOGLE, J. M., CHOW,M. & FILMAN,D. J. (1985). The three-dimensional structure of poliovirus at 2.9 .A. resolution.
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KEW, O. M. & NOTTAY,B. K. (1985). Evolution of the oral polio vaccine strains in humans occurs by both mutation
and intermolecular recombination. In Modern Approaches to Vaccines." Molecularand ChemicalBasis of Virus
Virulence and Immunogenicity, pp. 357-362. Edited by R. M. Chanock & R. A. Lerner. New York: Cold
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