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
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ABSTRACT |
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The sequences obtained in this study have been assigned GenBank accession nos AY323842AY323852.
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INTRODUCTION |
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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 12 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 20002001. 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.
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METHODS |
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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 L9, 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 24323477). 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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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).
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RESULTS |
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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 VP12A 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 VP12A junction region than in the entire VP1 gene, with the differences between individuals peaking at 4·8 % (Table 3).
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DISCUSSION |
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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 VP12A 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 (JuneJuly) did. Meanwhile, both synonymous and non-synonymous substitutions were found to accumulate in the VP1-coding gene, and the two later strains from SeptemberOctober 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 VP12A 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 VP12A junction region, but clustered together with the other JuneJuly 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 12 % 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
-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 VP12A 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.
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ACKNOWLEDGEMENTS |
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Received 21 July 2003;
accepted 29 October 2003.