Characterization of a rare natural intertypic type 2/type 3 penta-recombinant vaccine-derived poliovirus isolated from a child with acute flaccid paralysis

Polioviruses have three serotypes and are members of the human enterovirus C species that belong to the genus Enterovirus in the family Picornaviridae (Stanway et al., 2005). Polioviruses have a positive-sense, single-stranded polyadenylated RNA genome, which is one of the smallest genomes among viruses (7500 nt), enclosed in an icosohedral capsid comprising 60 copies of each capsid protein (VP4, VP2, VP3 and VP1). The viral RNA contains a long ORF flanked by a 5'-untranslated region (UTR) and a 3'-UTR. A single polyprotein translated from the RNA strand is first cleaved into three polyprotein precursors: P1, P2 and P3. P1 is processed to yield four capsid proteins: VP4 to VP1. P2 and P3 are the precursors of non-structural proteins 2A–2C and 3A–3D (Wimmer et al., 1993).

Polioviruses are among the most important and well-studied human pathogens, since they are the causative agents of acute paralytic poliomyelitis. To control the incidence of infection, the trivalent oral polio vaccine (OPV) containing three live attenuated strains of the poliovirus serotypes (Sabin 1, Sabin 2 and Sabin 3) has been widely used in the global polio eradication program. Although OPV is safe, problems have occurred due to its ability to revert from the attenuated phenotype. Consequently, the vaccine is associated with a low rate of paralytic poliomyelitis (vaccine-associated paralytic poliomyelitis; VAPP) (Dowdle et al., 2003). Further, polioviruses can also circulate invisibly without symptomatic patients in the population for several months and then revert from the attenuating form to a neurovirulent one (vaccine-derived polioviruses; VDPVs), causing outbreaks (Kew et al., 2002; Liang et al., 2006; Rousset et al., 2003; Shimizu et al., 2004; Yang et al., 2003).

In the WHO program for the global eradication of poliomyelitis (WHO, 2004a), VDPVs have been classified into three categories. (i) Circulating VDPVs (cVDPVs). These are associated with sustained person–person transmission. They represent the strains showing ≤99 % VP1 sequence homology to the ancestral Sabin OPV strains, and have caused paralytic cases in which related but non-identical viruses have been isolated (Kew et al., 2004). (ii) Immunodeficiency VDPVs (iVDPVs). These represent strains with ≤99 % VP1 sequence homology to the ancestral Sabin OPV strains, and are known to be excreted for a prolonged period from the same immunodeficient patient (Bellmunt et al., 1999; Kew et al., 1998). (iii) Ambiguous VDPVs (aVDPVs). These include other VDPVs that cannot be classified into the above two VDPV categories. These are viruses that either have been isolated from a single patient without immunodeficiency or are environmental isolates with an unidentified source (Kew et al., 2005). Two genetic characteristics, nucleotide mutations and genetic rearrangements, seem to underlie the occurrence of poliomyelitis outbreaks associated with cVDPVs (Kew et al., 2002, 2004, 2005).

While the genetic variability of polioviruses is mostly due to nucleotide substitutions resulting from a high error frequency during the replication of the viral RNA (Freistadt et al., 2007), genetic changes in polioviruses can also occur during the molecular genomic rearrangements that happen during virus replication (Cuervo et al., 2001). Poliovirus genomic rearrangement frequently takes place through homologous RNA recombination, mainly in the non-structural coding regions of the viral genome. Trivalent OPV permits co-infection of human gut cells with the Sabin 1, Sabin 2 and Sabin 3 strains, creating ideal conditions for intertypic recombination events. Analysis of VAPP infection cases revealed poliovirus recombination with a 50 % involvement of Sabin 2 strains and 67 % of Sabin 3 strains, while Sabin 1 strains were rarely involved (Furione et al., 1993). Most crossover sites of the type 2 recombinants (S2/S1 and S2/S3 recombinants) lie in the P3 coding region, and most crossover sites of type 3 recombinants (S3/S1 and S3/S2 recombinants) are located in the P2 coding region (Cuervo et al., 2001; Karakasiliotis et al., 2004).

In this study, we describe a natural intertypic type 2/type 3 penta-recombinant VDPV isolated in 1997 from a patient with acute flaccid paralysis (AFP) during virological surveillance in China. Primary characterization of the isolate revealed that this VDPV has uncommon genetic rearrangements which include a crossover site in the VP1 capsid genomic region. This observation led us to study the primary structure of the crossover sites and the genetic and phenotypic properties of this unusual poliovirus recombinant.

Primary characterization of strain CHN1025
Strain CHN1025 was completely neutralized with polyclonal antisera specific for type 2 but could not be neutralized with antisera for types 1 and 3. Thus, it was identified as a type 2 poliovirus. Intratypic differentiation (ITD) tests were performed by using two different methods. The PCR-RFLP ITD test revealed atypical Sabin 2 restriction patterns, and in the ELISA ITD test, the isolate was identified as SL, which indicated that it reacted with only Sabin 2-specific cross-absorbed rabbit antisera.

The entire VP1 coding sequences of strain CHN1025 revealed an uncommon genomic intertypic (type 2/type 3) recombinant structure, with a crossover site within the VP1 capsid coding region. The last 31 nt at the 3' end of the VP1 coding region were found to have high similarity (2 nt substitutions) with the Sabin 3 strain, while the rest of the VP1 coding region had high similarity with the Sabin 2 strain (8 nt substitutions). These similarities indicated its VDPV genomic features (a total of 10 nt substitutions in 903 nt in the entire VP1 coding region; homology with the relative Sabin strains was 98.9 %) and revealed that a crossover site was located in the VP1 coding region (Table 1).

Table 1. Nucleotide and amino acid substitutions in the penta-recombinant virus strain CHN1025 (GenBank accession no. AY948201) compared with Sabin 2, Sabin 3 and Sabin 1 Nucleotide and amino acid positions are numbered according to Sabin 2 (AY184220). Nucleotide or amino acid positions described as being involved in Sabin 2 attenuation are in bold. There are 10 nucleotide substitutions in the entire VP1 coding region; the first eight are located before the crossover site within the VP1 coding region and the other two are located after the crossover site.

Penta-recombinant structure of the CHN1025 genome
Comparing the complete genomic sequence of strain CHN1025 (7440 nt) with that of the reference Sabin 2 strain (GenBank accession no. AY184220[GenBank] ) revealed that at least five successive rounds of recombination events occurred in the genome in the VP1 capsid coding region and 2C, 3C (twice) and 3D^pol non-capsid coding regions during its evolution.

Strain CHN1025 was a type 2/type 3 penta-recombinant VDPV with uncommon genetic rearrangements which include a crossover in the capsid genomic region. Based on its genomic organization, it is characterized as a S2/S3/S1/S3/S1/S3 penta-recombinant, and the complete genomic sequence revealed the presence of five crossover sites. The first apparently occurred from an S2/S3 recombination event, with the crossover site located between nt 3354 and nt 3356 in the VP1 coding region, while the second crossover site was from an S3/S1 recombination event located between nt 4888 and nt 4895 in the 2C coding region. The third and fourth crossover sites were from S1/S3 and S3/S1 recombination events, with the two crossover sites located in nt 5447–5493 and nt 5671–5690 in the 3C coding region, respectively. The last crossover site was from another S1/S3 recombination event located between nt 6064 and nt 6092 in the 3D^pol coding region (Fig. 1).

(43K): Fig. 1. Schematic genomic structure of CHN1025. (a) The primer pairs for two long distance PCR amplifications to obtain the whole genome sequence of CHN1025. (b) The five proposed crossover sites are shown in detail. The last nucleotides, differentiating the sequence of CHN1025 from the 3' partner reference sequences, and the first nucleotides, differentiating the sequence of CHN1025 from the 5' partner reference sequence, i.e. the crossover sites, are highlighted by black shading. The filled circles and open circles above and below the nucleotide sequences, respectively, indicate the same and different bases between the sequence of CHN1025 and the partner reference sequences.

Replacement of attenuating determinants by back mutation
The complete genomic sequence of strain CHN1025 showed that its genome was collinear with that of the Sabin 2 strain, except for an insertion U at nt 96 in the 5'-UTR, suggesting that recombination did not alter the total number of capsid codons. A total of 37 nt substitutions were distributed throughout the genome, but most of them were located in the 5'-UTR (10 mutations out of total 748 nt; 1.34 %) and P1/capsid-coding region (17 mutations out of total 2637 nt; 0.64 %) (Table 1). The five crossover sites divided the genome into six parts, and most of the nucleotide substitutions (25 mutations out of total 3357nt; 0.74 %) were located in the first part of the original sequence; a small portion (12 mutations out of 4083 nt; 0.29 %) were present in the five parts of the donor sequences. The donor sequences of Sabin 1 between the second and the third crossover sites, and Sabin 3 sequence between the third and the fourth crossover sites contained no nucleotide substitutions compared with the relevant sequences of the relative Sabin strain (Table 1).

The nucleotide substitutions that had been identified as the principal determinants of the attenuated phenotype of the Sabin 2 strain had reverted through a transition A-to-G reversion at nt 481 in the 5'-UTR and a transition U-to-C reversion at nt 2909 in the VP1 coding region, leading to a Thr-to-Ile amino acid substitution of residue 143 in VP1 in isolate CHN1025 (Minor et al., 1993; Ren et al., 1991) (Table 2). Both replacements restored the consensus residues for the prototype wild-type 2 poliovirus strain MEF-1/EGY/1942.

Table 2. Neurovirulence of isolate CHN1025 in PVR-Tg21 transgenic mice ND, Not done.

Antigenic properties of the penta-recombinant VDPV
Antigenic analyses performed by the ELISA ITD method with cross-absorbed antisera characterized the type 2 isolate CHN1025 as a Sabin-like virus. The amino acid sequences within or near the predicted neutralizing antigenic (NAg) sites (Minor, 1990) were aligned with the penta-recombinant VDPV, Sabin 2 strain, two Egypt type 2 cVDPV strains (GenBank accession nos AF448782[GenBank] and AF448783[GenBank] ) (Yang et al., 2003), two Madagascar type 2 cVDPV strains (GenBank accession nos AM084223[GenBank] and AM084225[GenBank] ) (Rakoto-Andrianarivelo et al., 2007), two Spain type 2 iVDPV strains (GenBank accession nos EU566941[GenBank] and EU566950[GenBank] ) (Avellón et al., 2008) and the prototype wild-type 2 poliovirus strain MEF-1/EGY/1942. There were no amino acid substitutions in the NAg sites among the penta-recombinant VDPV, which coincided with the results of ELISA ITD (Fig. 2), and no amino acid changes were introduced in the VP1 capsid coding region as a consequence of the observed recombination (Table 1). Sabin-specific epitopes were present in strain CHN1025, suggesting that the virus was antigenically indistinguishable from the Sabin 2 reference strain. Although none of the amino acid substitutions are located in the NAg sites described so far, some are exposed on the virion surface, including residue 143 of VP1 which is in loop D–E (Lentz et al., 1997).

(7K): Fig. 2. Alignment of amino acid residues of NAg sites 1 (VP1, 88–106), 2 (VP2, 163–172; VP2, 268–270; VP1, 222–227), 3a (VP3, 54–61; VP3, 70–74; VP1, 287–292) and 3b (VP2, 71–73; VP3, 75–80) for Sabin 2, CHN1025, Egypt type 2 cVDPV strains (GenBank accession nos AF448782[GenBank] and AF448783[GenBank] ), Madagascar type 2 cVDPV strains (GenBank accession nos AM084223[GenBank] and AM084225[GenBank] ), Spain type 2 iVDPV strains (GenBank accession nos EU566941[GenBank] and EU566950[GenBank] ) and the prototype wild-type 2 poliovirus strain, MEF-1/EGY/1942. The PD50 values of Egypt type 2 cVDPV strains are taken from a report by Yang et al. (2003).

Neurovirulence of isolate CHN1025 in PVR-Tg21 transgenic mice
Isolate CHN1025 was isolated from a patient with AFP, demonstrating their neurovirulence for humans under natural conditions. The neurovirulence of isolate CHN1025 was examined in PVR-Tg21 transgenic mice expressing the human receptor for poliovirus and compared with the neurovirulence of Sabin 2 and the prototype wild-type 2 MEF-1/EGY/1942 strain. Under the assay conditions, MEF-1/EGY/1942 showed maximum neurovirulence, and the virus titre that induced paralysis or death in 50 % of the inoculated mice (PD₅₀) was 2.2, while isolate CHN1025 partially regained the neurovirulence characteristics of MEF-1/EGY/1942, as determined by PD₅₀ measurements, with a PD₅₀ value of ≥4.7 (Table 2).

Estimated evolution time of the capsid penta-recombinant
The approximate duration of replication of the capsid penta-recombinant VDPV after the initiating vaccine dose was estimated from the P1/capsid sequence difference between isolate CHN1025 and the Sabin 2 strain. The corrected proportion of synonymous substitutions (K_s) was 1.35 % of synonymous sites in the P1/capsid region (not including the donor recombinant sequences) and that of total substitutions (K_t) was 0.58 %. Under the assumption of constant nucleotide substitution rates of 3.2 % synonymous substitutions per synonymous sites per year and 1.1 % total substitutions per site per year in the P1/capsid region (Jorba et al., 2008), we estimated that the age of the capsid penta-recombinant VDPV was 154 days (from the K_s estimate) and 192 days (from the K_t estimate), respectively (Fig. 3). By comparing the calculated range of the estimation of initiating OPV dose with the epidemiological data, we estimated that the five successive rounds of recombination events most likely occurred between late July 1996 and early February 1997 (Fig. 3).

(20K): Fig. 3. Time line showing the date of birth of the patient with AFP, the dates of the subsequent sub-national immunization day (sNID; the day of an intensive immunization campaign when all children under 5 years old are targeted to receive vaccination against polio) in Shandong Province, the date of onset of AFP, the date of sampling and the estimated dates of the OPV dose that initiated VDPV replication.

In this paper, we describe an intertypic type 2/type 3 penta-recombinant poliovirus (strain CHN1025) isolated from a patient with AFP in the Shandong Province of China; the first crossover site in this virus was located in the VP1 coding region. Because this patient did not receive any dose of OPV after birth, the illness was caused by a poliovirus variant infection. Strain CHN1025 was identified as a type 2/type 3 penta-recombinant poliovirus with five crossover sites in its genome, which include a crossover site in the VP1 capsid genomic region, and most of the characteristics of the type 2 vaccine strain, such as antigenic properties in ELISA ITD with cross-absorbed antisera (van der Avoort et al., 1995), the same NAg sites and moderate neurovirulence. Despite these similarities, nucleotide sequencing showed a 1.1 % nucleotide variation in the VP1 coding region, so strain CHN1025 was identified as a VDPV. According to the WHO program of global eradication of poliomyelitis (WHO, 2004a), this VDPV is undoubtedly an aVDPV, because it does not have the genetic characteristics of iVDPV, such as numerous amino acid changes in NAg sites (Bellmunt et al., 1999; Kew et al., 1998).

To the best of our knowledge, this is the first report of a type 2/type 3 penta-recombinant VDPV which includes a crossover site in the VP1 capsid coding region. Recombination among OPV strains is easily detectable because the sequences of the parental vaccine strains are well defined (Toyoda et al., 1984). Natural recombination in polioviruses was first recognized when viruses with chimeric non-capsid sequences were isolated from children exposed to the OPV (Cammack et al., 1988). Although studies of polioviruses isolated from patients with poliomyelitis have demonstrated a high frequency of genetic recombination (Guillot et al., 2000), the recombinant polioviruses described here are unusual because they have five crossover sites including a crossover site within the VP1 capsid coding region of their genome, while crossover sites in the intertypic recombinants excreted by children exposed to OPV are usually restricted to the P2 or P3 non-capsid region (Cammack et al., 1988; Cuervo et al., 2001). No natural type 2/type 3 intertypic recombinants with a crossover site in VP1 coding region have been reported previously, and only few natural intertypic capsid-recombinant polioviruses have been reported. All reports have been on type 3/type 2 intertypic recombinants (Blomqvist et al., 2003; Dedepsidis et al., 2008; Martín et al., 2002). Natural intertypic recombination with a crossover site in the capsid coding region is a rare phenomenon, and no amino acid changes are introduced in the VP1 capsid region as a consequence of the observed recombination, possibly due to structural constraints that exist to maintain the integrity of the capsid shell. The integrity of the capsid region of poliovirus seems to be very important for propagation of the viruses themselves (Kohara et al., 1988).

A small portion of the nucleotide substitutions located in the donor sequences, two in particular, contained no nucleotide substitutions, suggesting that the donor sequences were relatively new. At least five successive rounds of recombination events occurred in the CHN1025 genome over a relatively short time period. This view was reinforced by the estimates of the apparent viral infection, which indicated that this occurred within 154 days (from the K_s estimate) and 192 days (from the K_t estimate) before the sampling dates. This is about the time of two rounds of mass immunization campaigns in Shandong Province, so there is a possibility that the virus was acquired by the patient from contact with a vaccine recipient, but unfortunately there is no way to test this possibility in this case, since additional stool specimens were not available from the patient's close contacts. The VDPV may infect the patient later in life, possibly close to the time of paralysis, and the exact source of the VDPV was unknown, as routine OPV coverage was high in Shandong Province in 1997 (Fig. 3).

The result of a neurovirulence test for isolate CHN1025 performed on PVR-Tg21 transgenic mice showed that MEF-1 virus is significantly more neurovirulent than strain CHN1025. Even though nucleotide substitutions at positions 481 of the 5'-UTR and 2909 of VP1 coding region (aa 143), regions known to be neurovirulence determinants, were present in the genome, moderate neurovirulence was observed in the transgenic mice. This observation may have been because the neurovirulence determinants of type 2 polioviruses do not strictly correlate with those in transgenic mice (Buttinelli et al., 2003).

Because the base substitutions contributing to the attenuated phenotype of the Sabin 2 strain readily revert during replication in the human gut, the viruses excreted by healthy vaccinees are frequently less attenuated than the original OPV strains (Macadam et al., 1993). Reversion is very rapid for the Sabin 2 strain because attenuation is primarily determined by only two highly unstable substitutions (Minor et al., 1993; Ren et al., 1991). The intense selection against the attenuating mutations suggested that the revertants replicate more efficiently in the human gut (Minor & Dunn, 1988). Therefore, it appears likely that isolate CHN1025, a Sabin 2 revertant, with increased potential for neurovirulence and transmissibility, is selected in communities where OPV is used, such as in Shandong Province. However, their spread in Shandong Province was restricted by the high population immunity (Yang et al., 2003).

It is known that the type 2 OPV strain spreads to unvaccinated children more easily than the other two serotypes (Fine & Carneiro, 1999). Therefore, this biological feature would favour its divergence and emergence as a paralysis-causing pathogen to unvaccinated children. Determining whether neurovirulence and transmissibility of VDPVs could be the result of the recombination remains to be elucidated. Indeed, VDPVs will become increasingly important as the prevalence of wild polioviruses decreases and OPV becomes the only remaining source of poliovirus infection, and as VDPVs can emerge in any country that uses OPV with insufficient vaccine coverage. The recent VDPV outbreaks also highlight the importance of maintaining sensitive poliovirus laboratory surveillance. Such surveillance will have major implications for the cessation of immunization with OPV after it is certified that wild polioviruses have been eradicated.

Patient history and clinical specimens.
A type 2 poliovirus recombinant (strain CHN1025) was isolated from a 7-month-old boy from Chiping County (population: ∼570 000) of Shandong Province, China, who developed clinical symptoms of muscular weakness, hypotony and paralysis on 3 February 1997. The patient was not known to have ever received a dose of OPV after birth and showed no signs of immunodeficiency at the time of presentation (data not shown). Two stool specimens were collected from the patient at a 24 h interval on 6–7 February 1997. He had residual paralysis at the 60 day clinical follow-up and his condition was classified as AFP by the Shandong provincial and National polio diagnosis experts group.

Viral isolation and primary identification.
Human rhabdomyosarcoma and L20B (a mouse cell line carrying the human poliovirus receptor) cell lines were used to isolate viruses from the stool specimens by standard methods (WHO, 2004b). Isolates were initially characterized by a micro-neutralization assay using poliovirus type-specific rabbit polyclonal antisera [National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands] (WHO, 2004b). ITD was performed using the PCR-RFLP (Balanant et al., 1991) and ELISA (van der Avoort et al., 1995) methods in order to investigate the wild or vaccine origin of the poliovirus isolate.

Viral RNA extraction and reverse transcription.
Viral RNA was extracted from the viral isolate using a QIAamp viral RNA mini kit (Qiagen) and stored at –80 °C until further use. SuperScript II RNase H^– reverse transcriptase [1 µl (200 U); Invitrogen] was used to produce single-stranded cDNA from 5 µl purified viral RNA. The cDNA synthesis was primed by using primers 7500A and Q8 (Fig. 1a; Supplementary Table S1, available in JGV Online), respectively, and performed at 42 °C for 2 h, followed by 60 °C for 15 min to inactivate the enzyme. Finally, RNA in an RNA : DNA hybrid was specifically degraded with 1 µl RNase H (Promega) at 37 °C for 30 min.

Full-length genome amplification.
Two long-distance PCR amplifications were performed by using the TaqPlus Precision PCR system (Stratagene), which consists of a blend of Stratagene cloned Pfu DNA polymerase (proof-reading) and Taq2000 DNA polymerase (non-proof-reading). Reactions contained 5 µl cDNA (see above), 0.1 mM each dNTP, 10 µl TaqPlus buffer, 1.0 ng µl^–1 forward (0001S48 or Y7) and reverse (Q8 or 7500A) primers (Fig. 1a; Supplementary Table S1) and 5 U TaqPlus enzyme in a 100 µl reaction. The amplification was carried as follows: 30 cycles of 94 °C (30 s), 60 °C (30 s) and 72 °C (6 min), followed by 94 °C (1 min) and 72 °C (20 min).

Nucleotide sequencing.
Two long-distance PCR products were purified using a QIAquick gel extraction kit (Qiagen). Cycle sequencing reactions were carried out using the version 3.0 of the BigDye terminator chemistry (Applied Biosystems), using the primers listed in Supplementary Table S1. Sequencing was performed in both directions using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), and every nucleotide position was sequenced at least once from each strand. The 5' segment sequences were determined by using the 5' rapid amplification of cDNA ends (RACE) core set (TaKaRa Biomedicals) according to the manufacturer's instructions.

Location of the crossover sites.
The sequences of the isolates were aligned with the reference strains by using MEGA program v4.0 (Sudhir Kumar, Arizona State University, Arizona, USA) (Tamura et al., 2007); the resulting reference strain sequences were found to be sequences with the GenBank accession nos AY184219[GenBank] , AY184220[GenBank] and AY184221[GenBank] for Sabin 1, Sabin 2 and Sabin 3, respectively. Plots of nucleotide similarity were created using SimPlot program v3.5.1 (Stuart Ray, Johns Hopkins University, Baltimore, Maryland, USA) (Lole et al., 1999). The crossover sites were identified as being located between the last nucleotide, differentiating the clinical sequence from the 3' partner reference sequences, and the first nucleotide, differentiating the clinical sequence from the 5' partner reference sequence.

Neurovirulence testing in PVR-Tg21 transgenic mice.
A neurovirulence test was carried out using PVR-Tg21 mice that expressed the human poliovirus receptor (CD155) (Georgescu et al., 1997). The type 2 reference Sabin attenuated strain [obtained from the National Institute for Biological Standard and Control (NIBSC), UK] and prototype wild-type 2 neurovirulent MEF-1 strain [obtained from the National Institute of Infectious Diseases (NIID), Japan] were used as virus controls in the test. In brief, six mice (4-weeks-old; three males and three females) were inoculated intracerebrally with 30 µl each virus dilution [in 10-fold increments; ranging from 2.5 to 6.5 log 50 % cell culture infective dose (CCID₅₀) per mouse]. The mice were examined daily for 14 days after inoculation, and the number of paralysed or dead mice was recorded. The virus titre that induced paralysis or death in 50 % of the inoculated mice (PD₅₀) was calculated by using the Kärber formula (Kärber, 1931) and expressed as PD₅₀ per mouse.

Estimating the date of the initiating OPV dose.
The date of the initiating OPV dose for the AFP case patient described in this study was estimated from the K_s (synonymous substitutions per synonymous site) and K_t (total substitutions per site) values by assuming evolution rates of 0.032 synonymous substitutions per synonymous site per year and 0.011 total substitutions per site per year (Jorba et al., 2008).

Nucleotide sequence accession number.
The complete genomic sequence of the natural intertypic type 2/type 3 capsid penta-recombinant VDPV described in this study was deposited in the GenBank database under the accession number AY948201.

We thank Dr. Chen-Fu Yang and Dr. Kew Olen for kindly sharing some nucleotide sequences for primers. This study was supported by the National Key Technology R&D Program of China (Project no. 2008BAI56B00), National Key Science and Technology Projects of China (Project no. 2008ZX10004-008) and grant I8/181/978 from the World Health Organization.

Footnotes

^,†, Haiyan Wang² †These authors contributed equally to this work.

The GenBank/EMBL/DDBJ accession number for the complete genomic sequence of the natural intertypic type 2/type 3 capsid penta-recombinant VDPV sequence is AY948201.

A supplementary table of primer sequences is available with the online version of this paper.