Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months

Tapani Hovi1, Noora Lindholm1, Carita Savolainen1, Mirja Stenvik1 and Cara Burns2

1 Enterovirus Laboratory, Department of Microbiology, National Public Health Institute (KTL), Mannerheimintie 166, 00300 Helsinki, Finland
2 Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA

Correspondence
Tapani Hovi
tapani.hovi{at}ktl.fi


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Wild-type 1 poliovirus (wtPV1) strains were isolated from two young healthy brothers shortly after arrival in Finland from Somalia in 1993. Twelve (sibling A) and 18 (sibling B) specimens collected over a period of more than 6 months yielded wtPV1. Partial sequences obtained from the one and two earliest isolates from sibling A and B, respectively, were nearly identical, differing from each other by only one or two nucleotides. Subsequently, the virus evolved separately in both siblings so that maximal differences between strains derived from a given subject peaked at 2·2 % for sibling A, at 1·5 % for sibling B and at 2·5 % between the two siblings in the VP1-coding part of the genome. All substitutions in the 150 nt VP1–2A junction region were synonymous, whereas as many as eight of the 31 variable positions in the remaining VP1-coding region encoded amino acid replacements in at least one strain. Probable structural locations of the variable amino acid positions were mapped to the published PV1/Mahoney structural model. Most of the substitutions occurred around the fivefold axis in motifs that are known to be or suspected to be targets of neutralizing antibodies. We suggest that the striking genetic divergence observed between the strains was based on a combination of bottleneck transmission events and antigenic drift during the prolonged period of poliovirus replication.

The sequences obtained in this study have been assigned GenBank accession nos AY323842AY323852.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polioviruses (family Picornaviridae, genus Enterovirus) are small non-enveloped RNA viruses. Replication of the messenger-sense RNA genome is prone to errors due to frequent single base misincorporations and the absence of proofreading. As a consequence, the viruses exist as swarms of slightly divergent mutants (quasispecies), which is a critical feature for the striking capacity of adaptation of the viruses. Frequently occurring homologous recombination provides an additional mode of generating divergence among polioviruses and other enteroviruses (Domingo & Holland, 1997). Various selection pressures operate within the infected host individual and – together with bottleneck purification during poliovirus transmission between host individuals – bring about independent parallel replication lineages (Rico-Hesse et al., 1987; Mulders et al., 2000). These typically show characteristic sequence patterns, which are used for identification of the genetic source of a given virus strain and analysis of virus transmission routes. Synonymous base substitutions in a genetic continuum of virus isolates have also been used for analysis of fixation rates of neutral mutations and for calculating approximate dates of defined lineage origins and divergences (Kew & Nottay, 1984; Kinnunen et al., 1991; Gavrilin et al., 2000; Liu et al., 2000).

Poliovirus infection in humans is typically acute but may continue for a few months. In paralytic cases caused by the wild-type virus, virus titres in faeces and other excreta rapidly decrease during the few weeks following onset of disease (reviewed by Alexander et al., 1997). Hence, for diagnostic purposes, two independent faecal specimens should be collected not later than 14 days after onset of paralysis for a negative result to be reliable (Birmingham et al., 1997). However, in most cases excretion of poliovirus is assumed to continue for several weeks at a lower rate. The length of excretion of wild-type poliovirus by subclinically infected individuals is less well known but is likely to be similar to that of paralytic cases. This view is also supported by observations on recipients of the live oral poliovirus vaccine (OPV). OPV-derived strains are regularly excreted for 1–2 months (Alexander et al., 1997; Piirainen et al., 1999) and occasionally much longer (Minor et al., 1986). Some OPV recipients with severe deficiencies in humoral immunity tend to remain chronically infected. During prolonged replication, the vaccine virus almost invariably reverts its attenuated character and acquires neurovirulent properties. As a consequence, chronically infected individuals may present with paralytic disease some years after OPV administration (Kew et al., 1998) and may also transmit the reverted virus to their close contacts. This raises concern for the desired future global stopping of OPV immunization, which will be considered after the eradication programme has been completed. Immune-deficient individuals receiving OPV during the last wave of immunization may excrete the virus for years and spread it to newly born children who are no longer being vaccinated. Studies aimed at evaluating the proportion of immune-deficient OPV recipients who remain chronically infected are in progress. The potential risk caused by circulating OPV-derived viruses to non-immune individuals was demonstrated in the Hispaniola outbreak in 2000–2001. OPV-derived type 1 poliovirus was readily spread in the poorly vaccinated population and was able to cause more than 20 paralytic cases (Kew et al., 2002; WHO, 2000). Smaller outbreaks of a similar kind have occurred more recently in the Philippines (MMWR, 2001) and Madagascar (WHO, 2002).

A few reports have been published on the extensive genetic divergence generated during chronic infection with OPV-derived strains lasting for several years (Yoneyama et al., 1982; Kew et al., 1998; Bellmunt et al., 1999; Gavrilin et al., 2000; Martín et al., 2000). Much less is known about the occurrence and features of long-term excretion of wild-type poliovirus. We have had the opportunity to characterize a set of wild-type 1 poliovirus (wtPV1) strains isolated from two young healthy brothers emigrating to Finland from Somalia in 1993. In this paper we present an analysis of partial genomic sequences of the virus strains that were excreted by both children for as long as 6 months.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus excretors and faecal samples.
Wild-type 1 poliovirus strains were isolated in March 1993 from two healthy native Somalian brothers (hereafter referred to as sibling A and sibling B), aged 2·7 and 3·5 years, respectively, shortly after their arrival in Finland to rejoin their parents who had arrived earlier. The polio immunization history of these boys was not known. Children immigrating to Finland from poliovirus-endemic countries were usually screened for potential poliovirus excretion irrespective of their health condition. Immigrant children were rapidly adopted into the national immunization programme and given the missing vaccine doses in a facilitated schedule. In the case of polio immunizations, this meant a dose of inactivated poliovirus vaccine (IPV) at 0, 2 and 8 months. After the initial discovery of wild-type poliovirus excretion, the brothers were kept at their home with a minimal number of visitors. Inactivated poliovirus vaccine was used to complete any deficiencies in the vaccination schedule of contacts. To monitor continuation of virus excretion, repeated faecal samples were collected, initially biweekly and, later on, monthly. Altogether 23 samples were examined from each sibling over a total follow-up period of about 10 months between March 1993 and January 1994. After stopping poliovirus excretion, they were given three doses of IPV and, according to the regular programme in Finland, they are scheduled for booster doses of IPV at 6, 11 and 16 years. These two children have been the only persons found to excrete wild-type poliovirus in Finland since 1984.

Virus isolation, typing and intratypic differentiation.
Isolation and identification of virus from the faecal samples was carried out by standard techniques. Briefly, suspensions of approximately 10 % (v/v) faecal material were extracted with chloroform and inoculated in tube cultures of four continuous cell lines, GMK and Vero (both originating from African green monkey kidney), and A549 (derived from human bronchial carcinoma tissue) and L{alpha}9, a recombinant murine L cell expressing the human poliovirus receptor (Hovi & Stenvik, 1994). Three tubes of each cell line were used per sample (sample dilutions 1 : 1, 1 : 2 and 1 : 10). Virus growth was monitored by daily microscopy, and enterovirus-like cytopathic agents were identified by in-house cross-secting pools of enterovirus antisera (LBM type). For suspected adenovirus isolates, a latex-agglutination kit exploiting group-specific hexon antisera (Adenolex; Orion Diagnostica) was used. The identity of poliovirus strains was confirmed by neutralizing monotypic rabbit antisera. For the purposes of this study, the remaining 19 original stool specimens stored frozen for about 7 years were thawed and 10 % suspensions were inoculated as above in L20B cells, another murine cell line expressing the human poliovirus receptor (Pipkin et al., 1993). For some specimens, additional cultures were made in GMK and HeLa cells (derived from human cervical carcinoma). The heterogeneity of virus strains in a given sample was also examined by inoculating freshly made faecal suspensions in L20B cells and using the plaque technique as previously described (Mulders et al., 1999).

Intratypic differentiation of the strains was carried out with two World Health Organization (WHO)-recommended methods (van der Avoort et al., 1995): antigenic characterization using polyclonal, absorbed antisera and RNA probe hybridization using Sabin strain-specific probes (De et al., 1995).

Partial genomic sequencing.
The viral RNA was isolated from 100 µl harvested cell culture (corresponding to approximately 105 cells) using the RNeasy Total RNA kit (Qiagen). Upon purification, RNA was eluted from the columns with 30 µl DEPC-treated distilled water and subsequently stored in aliquots at -70 °C. RT-PCR was used to amplify three partially overlapping genomic regions covering the entire capsid protein VP1-coding region and the N-terminal part of 2A (nt 2432–3477). The primers used in the RT-PCR are listed in Table 1. All PCRs were carried out in a PTC 100 Programmable Thermal Controller (MJ Research Inc.).


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Table 1. Primers used in generation of the sequenced amplicons

Nucleotide sequences are marked using standard IUB ambiguity codes: Y=T/C, R=G/A, W=A/T, M=A/C, N=A/G/C/T, I=deoxyinosine.

 
PCR products were purified using a PCR purification kit (QIAquick; Qiagen) in the case of a single band in the electrophoresis or, in the case of multiple bands, using a gel extraction kit (QIAquick, Qiagen). The purified products were used in cycle sequencing (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit; Applied Biosystems) with the same forward and reverse primers (5 µM) as used in the RT-PCR. To minimize possible sequence ambiguities arising from base misincorporation during in vitro amplification, duplicate amplicons were independently amplified from the PCR products and the forward and reverse sequences were determined on separate amplicons. The cycle sequencing programme was 30 cycles of 30 s at 94 °C, 15 s at 50 °C and 4 min at 60 °C. Upon completion, the reactions were cooled to 4 °C. The purified product of the sequencing reaction was resuspended on the same day that it was sequenced in a mixture of ABI Blue Dextran (ABI Prism) and deionized formamide, and run in an automated DNA sequencer (ABI Prism, model 377).

Sequence analysis.
Sequence data were analysed with Sequencing Analysis (version 3.1; ABI) and Sequence Navigator (version 1; ABI) for pairwise comparisons. Multiple sequence alignments were made using PILEUP, part of the GCG program suite (version 10, Genetics Computer Group) and ClustalX. Distance matrices were estimated using the DNADIST and PROTDIST programs, part of the PHYLIP (phylogeny inference) package (version 3.572c; Felsenstein, 1993), using the maximum-likelihood model of nucleotide substitution with default values for parameters. Identical branching was observed with the Kimura two-parameter model as well as with a transition/transversion ratio of 3·4 (estimated with Puzzle 4.0). Dendrograms were drawn using the neighbour-joining option in NEIGHBOR (PHYLIP) and were visualized using NJPLOT or TREEVIEW (version 1.5.3). Bootstrap analysis of the VP1 nucleotide sequences was performed using the SEQBOOT program of the PHYLIP package with 1000 replicates. Comparisons were made with previously published sequences (GenBank accession nos AF233098, AF233111, AF233112, AF233114, AF405615, AF405636, AF405640, AF405642, AF405647, AF405650, AF405653, AF405654, AF405655, AF405659, AF405661, AF528821).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Long-term excretion of wild-type 1 poliovirus by two healthy children
Two young brothers emigrating from Somalia were initially examined for potential excretion of poliovirus within a few days after arriving in Finland in 1993. Both were found to excrete wtPV1 in the very first specimen. This prompted a follow-up project where 23 stool specimens from each brother were collected at various intervals and examined for the presence of poliovirus. Twelve faecal samples from sibling A yielded poliovirus in cell culture extending as far as 7 months after the initial enrolment. The corresponding figures for sibling B were 18 samples and 7 months. All poliovirus isolates obtained were of serotype 1 and all examined isolates were shown to be non-Sabin-like in intratypic differentiation tests. Along with polioviruses, adenoviruses were isolated from some samples (Table 2). Sampling was stopped after two or three successive negative samples.


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Table 2. Survey of poliovirus excretion

Faecal specimens were inoculated in cell culture not later than 1 day after arrival in the laboratory and cytopathic viruses were identified by immunological techniques. The indicated virus was isolated in one or more cell lines. Polioviruses (PVs) were identified finally by monotypic rabbit antisera. Intratypic differentiation (ITD) was initially carried out for a proportion of specimens only. The PV1 strains that were not characterized in this way were treated as if they were wild-type. PV1 in bold means that the isolate was subjected to partial genomic sequencing in this study. All suspected adenoviruses were identified using the Adenolex kit; the adenovirus strain isolated from the AH specimen of 29 September 1993 was further identified as serotype 2. For other adenovirus strains, serotyping was not attempted. NSL, non-Sabin-like, probably wild strain; -, negative result.

 
The screening of the healthy children was initially started solely because of the immigrant status and during the follow-up no symptoms that could be associated with poliovirus infection were recorded. Because of the lack of any clinical symptoms, the hospital in charge and the parents agreed that there was no reason to make a venipuncture. Therefore, we could not examine possible poliovirus antibodies in the blood. Faecal samples from the parents were also examined some weeks after collecting the first poliovirus-positive sample from the children. No virus could be detected from these specimens in cell culture. No cases of confirmed or suspected poliomyelitis were reported in Finland in 1993 or subsequently. Likewise, regular environmental screening in the Helsinki region has not detected a single poliovirus strain since 1985 (Poyry et al., 1988; Hovi et al., 2001).

Genetic variation of poliovirus during replication in a given host
For the current sequencing studies, 19 specimens stored for 7 years at -20 °C were reinvestigated by inoculating freshly made suspensions into the poliovirus-selective L20B cells. Fourteen showed CPE typical of enteroviruses. Cultures remaining negative or showing atypical CPE were tested for adenovirus either directly or after subculturing in GMK and HeLa cells. An adenovirus was isolated from each of the remaining five specimens. In 16 out the 19 specimens, the results were identical with the initial results obtained 7 years earlier (not shown).

RNA was extracted from 14 L20B cell cultures inoculated with freshly made suspensions of the stored stool specimens. Genomic regions encoding VP1 and the N-terminal portion of the 2A protease were amplified in three separate RT-PCR reactions. One of the harvested cultures did not produce clear amplicons in spite of repeated attempts. Several wild-type PV1 isolates from both siblings (seven for A and six for B) were subjected to partial genomic sequence analysis. Three partially overlapping regions ranging from the 3'-terminal part of VP3 to the 5'-terminal part of the 2A protein gene were sequenced and the sequences obtained were aligned separately for the entire VP1 and for the traditional 150 nt VP1–2A junction region. Sequences obtained from the three early isolates (11-Mar-93B, 01-Apr-93A, 01-Apr-93B; coded according to the collection date) were almost identical, differing from each other by one or two nucleotides only. Subsequently, the virus evolved separately in both siblings so that maximal differences between strains derived from a given child peaked at 2·2 % for sibling A, at 1·5 % for sibling B and at 2·5 % between the two siblings for the entire VP1-coding part of genome. The maximum within- and between-host individual differences were greater in the VP1–2A junction region than in the entire VP1 gene, with the differences between individuals peaking at 4·8 % (Table 3).


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Table 3. Extent of divergence between the characterized poliovirus type 1 isolates

Extract from the distance matrices calculated between the strains in different genomic regions. Maximum values in each category are shown.

 
Variation of deduced amino acid sequences in capsid protein VP1
In spite of the relatively greater interstrain differences in the VP1–2A junction region, as compared with the VP1 gene, no amino acid differences were seen between the sequenced strains in the VP1–2A junction region. In contrast, several variable amino acid positions were recorded in VP1 (Tables 3 and 4) as well as a few in the C-terminal end of VP3 (not shown). The proportion of synonymous substitutions of all substitutions was 100 % in the 150 nt VP1–2A junction region, whereas in the VP1-coding region nucleotide changes at as many as eight of the total of 37 variable positions resulted in amino acid substitutions. Probable structural locations of the variable amino acid positions were mapped on the published PV1/Mahoney structural model (Hogle et al., 1985). Most of the substitutions occurred around the fivefold axis in motifs that are known or suspected targets of neutralizing antibodies (Table 4). Because no blood specimens were taken from the studied individuals, an attempt was made to demonstrate neutralizing antibodies in faecal specimens (Valtanen et al., 2000). All four extracts examined were, however, negative (data not shown).


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Table 4. Accumulation of amino acid substitutions at different positions in VP1

 
Phylogenetic analysis
Analysis of the evolutionary differences between the sequenced strains by the PHYLIP package revealed unequivocally that all strains characterized were monophyletic, belonged to the designated Eastern African (EAAF)-B genotype and that distinct lineages were generated in the two siblings. Subsequent isolates from polio patients in Somalia and neighbouring countries indicated that members of the same genotype have continued to circulate until very recently. NJ analyses carried out separately for the entire VP1 region and the VP1–2A junction (Fig. 1) demonstrated some differences between the two genomic regions. In the case of sibling A, distinct divergence of two separate lineages (‘June–July’ and ‘September–October’) from the initial isolates was seen in the VP1-coding sequences (Fig. 1b), while in the VP1–2A junction region only one of the branches (June–July) was evident (Fig. 1c) and the last isolate had a sequence identical to that of the first isolate. No discrepancies were seen among the strains derived from sibling B. Two additional isolates from sibling B were successfully sequenced in the VP1–2A region only. In the phylogenetic tree, these July–August strains were located close to another July strain (Fig. 1c). An attempt was made to demonstrate genetic heterogeneity in individual specimens (Huovilainen et al., 1988; Kinnunen et al., 1990) by isolating eight plaques from newly made faecal extracts of specimen 10-Jun-93A and characterizing them by partial genomic sequencing in the VP1–2A region. However, all studied plaques were identical (data not shown).



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Fig. 1. Neighbour-joining analysis of nucleotide sequences of poliovirus strains isolated from two Somalian brothers (A and B) after emigration to Finland in 1993. (a) VP1 gene of selected PV1/Finland ex-Somalia/1993 isolates (arrow) analysed with representatives of different PV1 genotypes. VDPV, vaccine-derived poliovirus. (b, c) Relationships between individual strains in the VP1-coding region (906 nt, b) and in the VP1–2A junction region (150 nt, c). Numbers at the branches in (b) represent bootstrap values of 1000 replicate analyses. Strains are coded according to the date of sample collection and the sibling A or B.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Poliovirus excretion is usually considered to last for a couple of months, although longer excretion has been occasionally described, even in apparently immunologically normal individuals. It is well known that in patients with defective humoral immunity, poliovirus and other enterovirus excretion may continue for several years (Kew et al., 1998). In the cases of extended excretion time, several single nucleotide substitutions and, in the case of trivalent OPV recipients, inter-serotypic recombinations have been frequently observed (Minor et al., 1986; Lipskaya et al., 1991; Furione et al., 1993). These genetic modifications have been interpreted to reflect the emergence of strains with improved fitness compared with the original attenuated vaccine strains. We have shown in this paper that excretion of wild-type poliovirus by healthy children may also continue for at least 6 months and is also associated with the accumulation of single nucleotide substitutions during replication within an individual host.

The total excretion time in the studied healthy siblings remained obscure as neither the timing of the onset nor that of the ending of excretion was absolutely clear, although it seems likely that the children had contracted the infection not long before arrival in Finland, since the first virus strains isolated from the two siblings were practically identical. Although two or three of the last studied samples for siblings A and B, respectively, were poliovirus negative, continuation of virus replication at a low level cannot be excluded, since in one of the siblings there had been previous ‘virus negative’ periods with two or even three successive negative specimens. Phylogenetic analysis suggested that the reinitiation of poliovirus excretion after these ‘breaks' reflected continuation of the original infection rather than reinfection by a new virus strain or cross-contamination in the laboratory by, for instance, specimens from the other sibling who was excreting the virus more regularly. The titre of virus in the specimens appeared to be relatively low, as only undiluted extracts yielded plaques (data not shown). This may also explain why not all results from 1993 could be reproduced 7 years later. The observed long excretion time in healthy individuals has important epidemiological implications and corroborates the need for several years of confirmatory follow-up times after the last cases of poliomyelitis before a given country or region can be declared polio-free.

The follow-up was stopped for these children as no signs were evident of spreading of the infection to close contacts or to the community. Because the studied children showed no symptoms associated with the documented poliovirus infection, no blood samples were collected and we could not analyse immunoglobulins or other general aspects of their humoral immunity. We were unable to demonstrate neutralizing antibodies in faecal extracts but this observation cannot be taken as evidence of any deficiency in humoral immunity, as the specimens had been frozen and thawed several times, which is known to expose faecal immunoglobulins to destructive proteases (Hovi et al., 1982; Valtanen et al., 2000). Furthermore, the absence of any symptoms in 3-year-old children living in a tropical developing country strongly argues against the possibility of an overt immunodeficiency. The specific reason, if any, for the exceptionally long excretion time remains obscure.

Partial sequences were analysed from seven and six isolates, respectively, from siblings A and B, representing the demonstrated excretion time of more than 6 months in the two siblings. Striking ranges of interstrain divergence could be demonstrated in both siblings, and this was especially true in sibling A in the VP1–2A junction region previously used for molecular epidemiology in the polio eradication programme (Rico-Hesse et al., 1987). Interestingly, in this individual the latest strains did not show divergence from the initial ones in this region, even though several of those isolated in the middle of the follow-up period (June–July) did. Meanwhile, both synonymous and non-synonymous substitutions were found to accumulate in the VP1-coding gene, and the two later strains from September–October showed a branch of their own in the phylogenetic tree composed from the VP1 gene sequences. In this case, the divergence from the initial specimens was also well supported by bootstrap analysis. Therefore, we believe that a sublineage with an unmodified VP1–2A junction region coexisted in the child throughout the entire follow-up period, including the time when more divergent strains represented the vast majority of the quasipecies composition. Indeed, a strain identical to the initial and final strains was isolated in July, in between isolating two variants in the same month, both before and after the invariant virus in the VP1–2A junction region, but clustered together with the other June–July strains in an analysis based on the VP1 gene. It could have represented a recombinant between the two sublineages. However, site-by-site analysis of the sequence alignment could not confirm this hypothesis (data not shown). Attempts to demonstrate different components of the quasispecies by plaque analysis failed, but this does not exclude this possibility, as a putative persisting minority may have been present at too low a level to be detected. This situation is also consistent with the view that poliovirus infection in a given host is composed of a series of consecutive and partially overlapping bursts of replication in defined, separated, locations in the gut-associated lymphoid tissues (Gavrilin et al., 2000). This enables bottleneck transmission events during replication of the virus within a single host and may result in the observed variation in the level of virus shedding and generation of distinct sublineages (Kinnunen et al., 1992).

Genetic differences between concurrent poliovirus strains belonging to a given lineage or genotype are frequently used as a basis of calculation for estimated timing of divergence of sublineages. In these calculations a constant rate of accumulation of synonymous substitutions (molecular clock) is assumed to exist, and for the gene of poliovirus VP1, rates of 1–2 % change year-1 have been proposed (Kew et al., 1998, 2002; Gavrilin et al., 2000). Because the majority of substitutions are usually synonymous, one might be tempted to use total evolutionary differences in the calculations. We saw total substitution rates exceeding 2·0 % within an individual and approaching 2·5 % between the two siblings within less than 3 months (e.g. between the July and late September specimens) indicating that epidemiological conclusions should be drawn with caution in individual cases. The observed relatively high proportion of non-synonymous substitutions only partially moderates this notion.

Antigenic evolution has not only been previously described in association with extended replication of OPV-derived poliovirus in vaccinees (Minor et al., 1986; Kew et al., 1998), but also for a wild-type virus in some situations (Huovilainen et al., 1987, 1988). We did not analyse the antigenic properties of the studied strains with monoclonal antibodies (mAbs), but concentration of the observed amino acid substitutions at and around the known antigenic sites is consistent with the view that immune surveillance had been involved in the enrichment of some of the sublineages. This view assumes that the healthy virus excretors were indeed immune competent in spite of the long excretion time. However, we cannot exclude a relative defect in the immune response in the studied children, because this situation might favour generation of antigenic variants. A low concentration of serum antibodies practically targeted to a single antigenic site may select resistant mutants in a similar way to mAbs (Hovi et al., 1995). Likewise, IgA-deficient children may remain free of serious symptoms of infectious diseases for years and yet show extended poliovirus excretion in the presence of protective concentrations of neutralizing serum antibodies (Savilahti et al., 1988). It is known that amino acid substitutions in the surface-exposed loops of picornavirus capsid proteins may emerge in the absence of immunological selection (Fares et al., 2001), possibly because of less stringent structural constraints in these regions than in the protein core forming the {beta}-barrel (Goldman et al., 1998). In any event, generation of amino acid substitutions close to antigenic sites in VP1, together with no substitutions in the VP1–2A junction region, is difficult to explain other than as a result of immune selection.

In conclusion, we have shown in this paper that wild-type poliovirus excretion may occasionally continue in apparently healthy children for more than 6 months, in some cases interspersed with virus-negative periods. Long-term excretion was associated with rapid generation of molecular and, probably, antigenic divergence, which might have epidemiological implications.


   ACKNOWLEDGEMENTS
 
This work was supported by World Health Organization TSA I8/181/526. O. M. Kew is acknowledged for comments and suggestions to the manuscript. We are grateful to Lina De and Jaume Jorba at CDC, Atlanta, GA, for providing unpublished sequences and to the staff of the Outpatient Clinic of the Aurora Hospital and the Department of Health, City of Helsinki, for collecting the original specimens.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 July 2003; accepted 29 October 2003.