1 Department of Microbiology, Enterovirus Laboratory, National Public Health Institute (KTL), Mannerheimintie 166, 00300 Helsinki, Finland
2 National Institute of Public Health, Oslo, Norway
Correspondence
Soile Blomqvist
soile.blomqvist{at}ktl.fi
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ABSTRACT |
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The GenBank accession number of the sequence reported in this paper is AF541919.
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INTRODUCTION |
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The World Health Organization (WHO)-coordinated international initiative of worldwide eradication of wild-type poliovirus has two cornerstones, intensive immunization with polio vaccine, including national immunization days, and systematic virological surveillance. Both the oral live attenuated vaccine (OPV) and the inactivated vaccine (IPV) consist of all three serotypes (Sabin & Boulger, 1973). The rate of evolution of polioviruses is extremely high and considered to be mainly due to the high frequency of errors in RNA synthesis, roughly 10-4 per base pair per replication cycle. The genetic diversity of poliovirus strains is exploited in molecular epidemiology, a key component of poliovirus surveillance (Rico-Hesse et al., 1987
), currently based on sequence analysis of the VP1 coding region of the genome. Strains isolated from patients with an epidemiological connection regularly show only limited sequence divergence, while those from unrelated cases usually differ much more (Rico-Hesse et al., 1987
). Genomic analysis over a wider range has revealed that recombination is a frequent phenomenon in polio vaccinees, including strains isolated from cases of vaccine-associated paralytic poliomyelitis (VAPP) (e.g. Gammack et al., 1989
; Lipskaya et al., 1991
; Furione et al., 1993
; Georgescu et al., 1994
, 1995
; Georgopoulou & Markoulatos, 2001
).
Vaccine/vaccine and vaccine/non-vaccine recombinants have been described in many studies (e.g. Guillot et al., 2000; Cuervo et al., 2001
). Recently, vaccine-derived polioviruses (VDPV) have been associated with three outbreaks of poliomyelitis. Type 2 VDPV circulated in Egypt for over 10 years (19831993) and was isolated from 32 cases (Centers for Disease Control and Prevention (CDC), 2001a
). An outbreak of poliomyelitis on the island of Hispaniola was associated with circulating type 1 vaccine-derived poliovirus (Kew et al., 2002
). In the Philippines, type 1 VDPV was involved in three poliomyelitis cases in 2001 (CDC, 2001b
). In each of these three outbreaks, the VDPV strains were recombinants in which most of the genomic region encoding the non-structural proteins was apparently derived from a non-polio enterovirus origin.
In natural intertypic recombinant poliovirus strains characterized so far, the recombination junctions have been almost without exception in the genomic region encoding non-structural proteins. Two special genomic arrangements have been described in the intratypic recombinants. In Romania, a type 2 vaccine/non-vaccine strain with a 5' recombination site at the beginning of the coding region was isolated from a lethal VAPP case (Georgescu et al., 1995). Type 1 wild-type/vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. All these type 1 recombinants had a 367 nt block of sequence derived from Sabin 1 spanning 115 3'-terminal nucleotides of the VP1 gene and the 5' half of the 2A gene in a wild-type poliovirus 1 environment (Liu et al., 2000
).
In this study, we describe a Sabin3/2/3 recombinant which was isolated from a faecal specimen from a healthy child 12 weeks after administration of the oral poliovirus vaccine. Primary characterization of the isolate revealed a chimeric Sabin 3/Sabin 2 capsid structure. This observation led us to study further the complete genomic sequence, antigenic properties and temperature sensitivity of this virus.
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METHODS |
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Virus isolation.
Poliovirus 3 was first isolated from a faecal specimen from a 5-year-old boy. The index case, his 2-year-old brother and his 9-year-old sister stayed in Sri Lanka for 8 months and received a dose of OPV on the national immunization day, 28 October 2000. They had all been previously immunized with inactivated poliovirus vaccine (IPV) in Norway according to the usual schedule, which includes three doses of IPV at 6, 8 and 16 months of age and two revaccinations at the ages of 78 and 15 years. During the last week in Sri Lanka, the index case got an unexplained fever and was hospitalized soon after returning to Oslo. He had several septic peaks every day, but no other symptoms. Both siblings were healthy.
Poliovirus 3 strain PV3/NOR/00/46220, from the index case, was isolated in L20B cells, typed as poliovirus 3 in the National Institute of Public Health, Oslo, Norway, and sent to the Regional Reference Laboratory [National Public Health Institute (KTL), Helsinki, Finland] for intratypic differentiation (ITD) tests on 16 January 2001. Approximately 12 weeks (83 days) after the OPV dose, poliovirus 3 strain PV3/NOR/01/8 was also isolated from the younger brother of the index case and at the same time, PV3/NOR/01/1 was isolated from a faecal specimen from the 9-year-old sister of the index case and sent to KTL on 6 February 2001.
Confirmation of serotype and ITD.
The PV3 isolates were analysed according to the protocol recommended by WHO. The serotype was confirmed by neutralization with polyclonal antisera [obtained from National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands]. ITD by enzyme immunoassay (EIA) was carried out with cross-absorbed antisera to PV3 Sabin and non-Sabin-like PV3 (RIVM). The molecular ITD was carried out by restriction fragment length polymorphism analysis of RT-PCR amplicons (RT-PCR-RFLP). A 480 nt genomic segment of the VP3/VP1 coding region was digested with three restriction enzymes, DdeI, HaeIII and HpaII (New England BioLabs). The patterns of the digests were analysed by agarose gel electrophoresis and compared with the patterns of the reference Sabin strains (Balanant et al., 1991).
RT-PCR and sequencing.
The entire VP1 protein coding region of PV3/NOR/01/8 was sequenced using two overlapping RT-PCR amplicons with the following primer sets: for the 5' part, primers 4548 and PV1A, and for the 3' part, PV2S and Q8 (Table 1). Both previously described and newly designed primers were used in the sequencing of the complete genome of the isolate PV3/NOR/01/8 (Table 1
). The protocols for RT-PCR and sequencing were the same for all primer pairs. Briefly, RNA was extracted from 100 µl of infected cell cultures with an RNeasy Total RNA Kit (Qiagen) according to the manufacturer's instructions. RNA (1 µl) was added to RT-PCR mixtures (total volume 50 µl) containing 67 mM Tris/HCl (pH 8·8), 17 mM (NH4)2SO4, 6 µM EDTA (pH 8·0), 200 µM each of dATP, dCTP, dGTP and dTTP (Roche Diagnostics), 1·5 mM MgCl2, 10 U RNasin Ribonuclease Inhibitor (Promega), 3·6 U avian myeloblastosis virus reverse transcriptase (Finnzymes), 2·7 U Thermoperfect Taq polymerase (Integro, Leuvenheim, The Netherlands) and 50 pmol each of the forward and reverse primers. The mixtures were incubated at 50 °C for 30 min and at 94 °C for 3 min. Thirty-five cycles of amplification (94 °C for 30 s, 42 °C for 30 s and 60 °C for 30 s) were followed by a final extension step at 60 °C for 5 min.
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Preparation of mouse antisera to the PV3/NOR/01/8 isolate.
The PV3/NOR/01/8 isolate was cloned by plaque purification in GMK cells. Five separate plaques were collected in 100 µl of Eagle's minimum essential medium supplemented with 2 % foetal calf serum. One plaque was passaged twice in GMK cells and the virus was purified by precipitation with polyethylene glycol and sucrose gradient centrifugation (Abraham & Colonno, 1984). The fractions were assayed by titration in RD cells; infective fractions were combined and concentrated by ultracentrifugation. The pellets were suspended in PBS; the protein concentration was measured and the virus preparation diluted to 51·8 µg protein ml-1.
Ten in-house-bred NIH mice (female, 8 weeks old) were used to produce antisera to the PV3/NOR/01/8 virus. Three inoculations of virus (each 5·18 µg per mouse) were given at 0, 2 and 4 weeks. The antisera were prepared from blood drawn 1 week after the last injection.
Micro-neutralization assay.
Neutralizing antibodies to Sabin 1, Sabin 2, Sabin 3 and PV3/NOR/01/8 were assayed by a standard micro-neutralization test. Fifty human serum specimens were selected from routine diagnostic material from the Department of Virology, University of Helsinki. Specimens had been collected during 1998 from healthy IPV-immunized children under 5 years of age. Twofold dilution series, starting from a 1 : 4 dilution, were made in duplicate in a 96-well plate from each human and mouse serum specimen. One-hundred TCID50 units of virus were added and the mixtures were incubated for 1 h (mice sera) or overnight (human sera) at 36 °C. After neutralization, 2x105 Vero cells were added to each well. The plates were sealed, incubated at 36 °C for 7 days and stained with crystal violet. The reciprocal of the dilution showing 50 % inhibition of virus-induced CPE was taken as the end-point titre.
Determination of neutralization indices.
Polyclonal antisera for Sabin 2 and Sabin 3 were diluted 1 : 20 and viruses Sabin 2, Sabin 3 and PV3/NOR/01/8 1 : 10. Equal volumes of virus and antiserum dilutions were mixed and incubated for 2 h at 36 °C for neutralization. The titres of remaining infectious viruses were determined in 96-well cultures of Vero cells at 36 °C. The neutralization indices of the sera were calculated as the logarithm of the ratio of the titre of the virus without antiserum to that with the tested antiserum.
Assay for temperature sensitivity.
Temperature sensitivity of the isolate PV3/NOR/01/8 was assayed in monolayer cultures of RD cells. Two 24-well plates were inoculated with 50 µl of undiluted virus stocks (Sabin 3, PV3/NOR/01/8 and PV3/Leon). After adsorption for 1 h at 36 or 40 °C, the unadsorbed virus inoculum was removed, maintenance medium was added and the plates were incubated at 36 or 40 °C. Wells were harvested at 8 and 24 h post-infection and total virus concentration was determined by the end-point dilution method in RD monolayer cultures in 96-well plates at 36 °C.
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RESULTS |
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To verify that the virus had a recombinant genome and was not a mixture of two distinct serotypes, it was plaque-purified. Five different plaques were collected and genomic sequences in the VP1/2A junction region determined. All five revealed the same recombination junction in the capsid coding region.
PV3 was also isolated, in Norway, from faecal specimens from the two siblings of the 2-year-old boy. The isolate PV3/NOR/00/46220 was typically Sabin-like in the ITD tests, while PV3/NOR/01/1 was confirmed as Sabin-like PV3 in an EIA test, but in RT-PCR-RFLP the virus gave an extra band with one of the restriction enzymes (HpaII). The sequence of the VP1-encoding gene of PV3/NOR/01/1 was 99·8 % identical to Sabin 3 without any sign of recombination. No further studies were performed with this virus.
Sequence analysis of the complete genome
The PV3/NOR/01/8 isolate was sequenced from nucleotide 11 to the 3' end of the genome. Two recombination junctions were identified (Fig. 1). The first recombination crossover point was in the VP1 capsid protein-coding gene from nucleotides 3274 to 3285. This recombination event led to the change in the genome from Sabin 3 to Sabin 2 and resulted in an insertion of six amino acid substitutions' in the carboxyl terminus of VP1 (amino acids 279, 286, 287, 288, 290 and 293 of VP1). The second crossover point, a change from the Sabin 2 genome to Sabin 3, was in the polymerase coding region, and included nucleotides 6824 and 6825 (numbering of Sabin 3).
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Ten mice were immunized with the PV3/NOR/01/8 virus. All mice produced neutralizing antibodies to both the immunogen strain and Sabin 3 (Table 4). Nine mice out of ten produced antibodies with slightly better neutralizing capacity to the PV3/NOR/01/8 than to Sabin 3. Sabin 1 and Sabin 2 were not neutralized by the immune sera.
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Temperature sensitivity
The PV3/NOR/01/8 virus was compared to Sabin 3 and the parental PV3/Leon strain as regards replication capacity at an elevated temperature (40 °C). Sabin 3 was temperature sensitive as expected while in the case of the PV3/NOR/01/8 strain a definitely lesser effect was seen (Table 5).
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DISCUSSION |
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The virus strain characterized was not isolated from a patient with suspected poliomyelitis but from a healthy child, who had been examined because of having visited a region that was considered to be a risk area for wild-type poliovirus circulation. In fact, Sri Lanka has been free of wild-type polio since 1993. Bearing in mind the recent outbreaks caused by circulating vaccine-derived poliovirus strains (cVDPV) (CDC, 2001a, b
; Kew et al., 2002
), careful analysis of the characteristics of poliovirus strains isolated from any location is recommended by WHO. Intratypic differentiation tests are performed on every poliovirus isolate to distinguish between vaccine and wild-type viruses. In this case, the preliminary molecular analysis (RT-PCR-RFLP) did not reveal any wild-type characteristics but, according to antigenic analysis (EIA), the isolate appeared to be on the borderline of Sabin-like and non-Sabin-like. The growth characteristics at elevated temperature supported the wild-type nature of this virus. Further sequencing revealed the novel composition of a Sabin recombinant poliovirus with a chimeric capsid protein VP1. While we were preparing the manuscript, we learnt that a similar recombinant virus with one of the recombination junctions within the VP1-coding region had also been observed and characterized by Martin et al. (2002).
Recombination is a frequent event in polioviruses, occurring at a rate of 10-4 per base pair per replication cycle at the RNA level (Jarvis & Kirkegaard, 1992). There may be a selective advantage for recombination to increase the fitness of the strain for replication in the human gut by preventing the accumulation of harmful mutations. Tripartite recombinant polioviruses with homotypic 5'- and 3'-terminal regions of type 3 have been described (e.g. Cuervo et al., 2001
). Usually, the first recombination site is in the P2 region and the second in P3, often within the polymerase gene. Structural constraints in the capsid proteins are considered to restrict enrichment of intertypic recombinants with chimeric capsid proteins. Indeed, the previously described recombinants in this category did not result in any amino acid substitutions. Survival of the current strain with a chimeric VP1 might be based on the fact that the inserted type 2-specific motif is located at the virion surface, allowing more freedom for aberrant folding. This was also consistent with the observation that the virus had altered type 3-specific antigenic properties but had not acquired any type 2-specific characteristics.
In our case, it seems likely but cannot be proven that the virus had been generated in the sampled vaccinee. The median divergence of the nucleotide sequence is 0·44 %, which is more than one would generally expect after 12 weeks of replication, but because of selection and chance, divergent lineages are occasionally generated rapidly (Kinnunen et al., 1991). We have previously proposed that many of these lineages may have a reduced fitness for transmission, as the divergence generated during a widespread epidemic did not strikingly exceed that seen between sequential specimens collected from a given individual (Huovilainen et al., 1988
; Kinnunen et al., 1991
).
Isolate PV3/NOR/01/8 had a nucleotide substitution of T for C at position 472 in the 5' non-coding region, which is a reversion to the sequence of the neurovirulent precursor of Sabin 3, P3/Leon/USA/1937. This substitution is found often in vaccine strains isolated from healthy vaccinees and VAPP cases, consistent with rapid reversion of this nucleotide in the human gut (Evans et al., 1985; Minor & Dunn, 1988
). Another sequence variation frequently associated with increased neurovirulence (nucleotide 2493) (Chumakov et al., 1992
) was not seen in our PV3/NOR/01/8 strain.
Three amino acid changes at known antigenic epitopes, in addition to the exchange of the C terminus of VP1, may explain why PV3/NOR/01/8 reacted as non-Sabin-like with cross-absorbed intratype-specific antisera. The mutations in VP2 at amino acid 165 and in VP3 at amino acid 77 were previously reported to be common both in healthy vaccinees and vaccine-associated poliomyelitis cases, possibly reflecting immune pressures in vaccinees (Macadam et al., 1989). The mutations in VP3, Ser-59
Asn and Asp-77
Asn, were also common in the Sabin 3 strains imported into The Netherlands. At least one of these mutations was seen in a set of Sabin 3 isolates which were double-reactive in the ITD assay (Reimerink et al., 1999
).
In spite of the altered antigenic epitopes, the serotype of the isolate was not altered, as shown by the virus being totally neutralized by polyclonal type 3 antisera and not at all by type 2 antisera. Despite the mutations in two of the four antigenic sites, the virus was readily neutralized with sera from IPV-vaccinated children. It seems that there is no risk of circulation of this virus in well-vaccinated populations.
Strain PV3/NOR/01/8 had almost entirely lost the temperature-sensitive phenotype of the parent Sabin viruses. The mutation that results in temperature sensitivity in type 3 Sabin virus involves the substitution of a phenylalanine for a serine residue at amino acid 91 in VP3 (Minor et al., 1989). This amino acid was Sabin-like in our virus. A number of Sabin 3 strains with reduced temperature sensitivity have been isolated from vaccine-associated cases. Besides amino acid 91 in VP3, eleven other amino acid substitutions have been suggested to be related to temperature sensitivity; among these is the mutation alanine for valine at position 34 in the VP1 capsid protein, also found in PV3/NOR/01/8.
In conclusion, we have characterized a recombinant poliovirus type 3 with a tripartite genome, in which the first recombination site was in the VP1 capsid region and the second in the polymerase gene. In spite of the amino acid changes in antigenic sites 2B and 3B, and the replacement of site 3A by Sabin 2-specific amino acids, the recombinant was neutralized by sera from IPV-vaccinated children as well as the parental Sabin 3 strain and did not acquire type 2-specific antigenic characteristics. Because of limited divergence from the parental Sabin strains, it seems that the recombinant virus had evolved in the vaccinee and does not represent a circulating VDPV.
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ACKNOWLEDGEMENTS |
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Received 12 July 2002;
accepted 29 October 2002.