Conceived and designed the experiments: YZ WX. Performed the experiments: YZ SZ DY GL RB DW LC HZ HA. Analyzed the data: YZ OK WX. Contributed reagents/materials/analysis tools: YZ. Wrote the paper: YZ.
Current address: Jilin Center for Disease Control and Prevention, Changchun, People's Republic of China
The authors have declared that no competing interests exist.
Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008.
Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3′ end of the
10 natural type 3/type 2 intertypic
Polioviruses, the causative agents of acute paralytic poliomyelitis, have three serotypes and are members of the human enterovirus C species of
The trivalent oral polio vaccine (OPV) contains three different poliovirus serotypes (type 1, 2, and 3). The use of OPV permits coinfection of the human gut cells with type 1, type 2, and type 3 vaccine strains, and thus providing favorable conditions for intertypic recombination. In fact, recombination is a very frequent phenomenon in poliovirus evolution and has been frequently found in patients with vaccine-associated paralytic poliomyelitis (VAPP)
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
In this study, we describe 10 different natural type 3/type 2 capsid-recombinant polioviruses isolated from nine acute flaccid paralysis (AFP) case patients and one healthy vaccinee during the virological surveillance period 2001–2008 in China. Primary characterization of these isolates revealed that the first crossover sites of these 10 isolates were all in the
All 10 virus isolates were completely neutralized with polyclonal antisera specific for type 3 but could not be neutralized with antisera for type 2. Thus, they were all identified as type 3 polioviruses. Intratypic differentiation (ITD) tests were performed by two different methods. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) ITD tests revealed typical Sabin 3 restriction patterns except for one strain (CHN6356) that showed an atypical non Sabin-like (NSL) pattern. In the enzyme-linked immunosorbent assay (ELISA) ITD tests, four isolates were identified as Sabin-like (SL), and two isolates (CHN5275 and CHN6053) were identified as double reactive virus (DRV), which indicated that they reacted with both Sabin 2-specific and type 2 wild poliovirus-specific cross-absorbed rabbit antisera. The results of ELISA ITD were not available for another four virus isolates due to the change of the ITD testing algorithm used in the laboratory (
Virus isolates | Accession numbers | Source |
Age(yr)/Sex | OPV history | Dates of | Intratypic differentiation |
|||
Last OPV | Onset | Sampling | PCR-RFLP | ELISA | |||||
CHN5275 | FJ859183 | AFP case | 0.8/F | 3 | 11-Jun-2001 | 8-Aug-2001 | 14-Aug-2001 | SL | DRV |
CHN6053 | FJ859184 | AFP case | 1.6/M | 3 | Unknown | 28-Jan-2002 | 26-Feb-2002 | SL | DRV |
CHN6060 | FJ859185 | AFP case | 0.7/M | 1 | 5-Dec-2001 | 24-Jan-2002 | 4-Feb-2002 | SL | SL |
CHN6213 | FJ859186 | AFP case | 0.8/M | 1 | Unknown | 20-May-2002 | 26-May-2002 | SL | SL |
CHN6218 | FJ859187 | AFP case | 1.5/M | 1 | 5-Dec-2001 | 29-Jun-2002 | 3-Jul-2002 | SL | SL |
CHN6356 | FJ859188 | AFP case | 0.4/M | 2 | 15-Aug-2002 | 6-Sep-2002 | 10-Sep-2002 | NSL | SL |
CHN11144 | FJ859189 | AFP case | 0.4/F | 1 | 16-Jun-2007 | 17-Jul-2007 | 27-Jul-2007 | SL | ND |
CHN11185h | FJ859190 | Healthy vaccinee | 1.0/M | 1 | Unknown | — | 1-Apr-2007 | SL | ND |
CHN12092 | FJ859191 | AFP case | 3.2/M | 3 | 16-Nov-2005 | 9-Sep-2008 | 9-Sep-2008 | SL | ND |
CHN12121 | FJ859192 | AFP case | 0.3/M | 1 | 1-Jul-2008 | 15-Aug-2008 | 22-Aug-2008 | SL | ND |
*AFP: Acute flaccid paralysis.
SL: Sabin like; NSL: non Sabin-like; DRV: Double Reactive Virus; ND: not done.
The entire
The yellow open rectangles indicated possible crossover sites.
Complete genomic sequencing of 10 natural type 3/type 2 intertypic vaccine-related poliovirus capsid recombinants with the first crossover sites within the
Virus isolate | Nucleotide and amino acid of neurovirulence determinants | Recombination pattern | Evolution of the viruses* | |||||
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||||||
nt472 | nt2034 | aa91 | nt2493 | aa6 | ||||
P3/Sabin |
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|
|
|
|
— | — | — |
CHN5275 | C |
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U | Ile | S3/S2/S3 | 0.34 (38 d) | 0.27 (88 d) |
CHN6053 | C |
|
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U | Ile | S3/S2/S3 | 0.56 (63 d) | 0.39 (127 d) |
CHN6060 | C |
|
|
U | Ile | S3/S2/S3 | 0.47 (53 d) | 0.24 (79 d) |
CHN6213 | C |
|
|
U | Ile | S3/S2/S3 | 0.35 (39 d) | 0.24 (79 d) |
CHN6218 | C |
|
|
U | Ile | S3/S2/S3 | 0.46 (52 d) | 0.24 (79 d) |
CHN6356 | C |
|
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U | Ile | S3/S2/S3 | 0.35 (39 d) | 0.20 (65 d) |
CHN11144 | C |
|
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U | Ile | S3/S2/S1/S2/S1 | 0.58 (65 d) | 0.36 (118 d) |
CHN11185h | C | C | Ser | U | Ile | S3/S2/S3 | 1.07 (120 d) | 0.36 (118 d) |
CHN12092 | C |
|
|
U | Ile | S3/S2/S3 | 0.46 (52 d) | 0.32 (105 d) |
CHN12121 | C |
|
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U | Ile | S3/S2 | 0.46 (52 d) | 0.20 (65 d) |
P3/Leon/37 | C | C | Ser | U | Ile | — | — | — |
Shaded area indicates the nucleotides and amino acids that were identical to the Sabin 3 strain, numbering according to the Sabin 3 strain (GenBank accession no: AY184221).
The results of the ELISA ITD method showed that strains CHN5275 and CHN6053 were two antigenic variants of the Sabin 3 strain; however, all 10 isolates were completely neutralized with polyclonal antisera to type 3 poliovirus but not with type 2 poliovirus antisera, according to the standard procedure
Interestingly, the NAg3a amino acid in the VP1 protein of type 3 wild polioviruses that were circulating throughout the world after 1980 were similar to that of the Sabin 2 strain, and the amino acid sequences were identical between VP1-286 and VP1-289. Type 3 wild polioviruses isolated from Angola and Tunisia also have the same residues of VP1-290, and type 3 wild polioviruses isolated from Nigeria, India, Pakistan, and Afghanistan, where wild polioviruses are still circulating, have a different residue at VP1-290 (from Thr to Ala). The neurovirulent precursor of Sabin 3 (Leon/USA/1937) also had a Lys residue at VP1-286 that was identical to that of the Sabin 2 strain (
The rectangle shows NAg3a displacement in VP1 from Sabin 3 to Sabin 2 due to the recombination. The dashed rectangle shows NAg3a of type 3 wild polioviruses circulated after 1980.
Eight of the ten
(a): Schematic genetic organization of Sabin 3 reference strain (GenBank accession number AY184221). The single open reading frame, flanked by
For each plot, the names of viruses of the query sequence were indicated in the bottom right corner, and for each bootscanning analysis, the names of viruses of the query sequence were indicated in the bottom left corner.
Strain CHN12121 was simpler in that it had only one crossover site located between nt3260 and nt3261 in the
Strain CHN11144 was a rare and complicated multi-recombinant poliovirus, and its genomic organization was characterized as a S3/S2/S1/S2/S1 tetra-recombinant. The complete genomic sequences revealed the presence of four crossover sites. The first was from a S3/S2 recombination event with the crossover site located between nt3233 and nt3240 in the
Although the numbers and positions of crossover sites were different among the 10
Although different lengths of nucleotides (55 to 136 nucleotides) were introduced in
All 10 VP1 capsid recombinants were compared to P3/Sabin strain as regards replication capacity at an elevated temperature (40°C), and showed different results of temperature sensitivities. P3/Sabin was temperature sensitive as expected with titer reduction of more than 2 logarithms at 36°C /40°C, and five VP1 capsid recombinants (CHN5275, CHN6060, CHN6218, CHN6356 and CHN12121) appeared similar results as P3/Sabin strain, while the property of temperature sensitivity of other five VP1 capsid recombinants showed a definitely lesser effect (titers reduced less than 2 logarithms at 36°C /40°C), this means that replication efficiency of them remains the same even at elevated temperatures (
Growth temperature and virus titer | ||||||
36°C | 40°C | Log titer reduction 36°C/40°C | ||||
Virus strain | 8 h p.i. | 24 h p.i. | 8 h p.i. | 24 h p.i. | 8 h p.i. | 24 h p.i. |
P3/Sabin | 6.500 | 8.250 | 3.250 | 3.750 | 3.250 | 4.500 |
CHN5275 | 8.500 | 8.875 | 7.000 | 6.750 | 1.500 | 2.125 |
CHN6053 | 7.625 | 8.250 | 7.500 | 7.500 | 0.125 | 0.750 |
CHN6060 | 8.375 | 8.875 | 6.875 | 6.625 | 1.500 | 2.250 |
CHN6213 | 7.875 | 8.125 | 6.750 | 6.750 | 1.125 | 1.375 |
CHN6218 | 7.750 | 7.875 | 6.125 | 5.625 | 1.625 | 2.250 |
CHN6356 | 8.000 | 7.875 | 6.125 | 5.625 | 1.875 | 2.250 |
CHN11144 | 8.250 | 8.750 | 7.125 | 7.375 | 1.125 | 1.375 |
CHN11185h | 8.500 | 9.000 | 7.125 | 7.125 | 1.375 | 1.875 |
CHN12092 | 8.500 | 8.500 | 7.250 | 7.125 | 1.250 | 1.375 |
CHN12121 | 8.000 | 8.375 | 5.750 | 5.125 | 2.250 | 3.250 |
*
The approximate evolution times of the polioviruses were estimated from the P1/capsid sequence differences between
Poliovirus is one of the first recognized viruses to undergo recombination
Some similar intertypic recombinant chimera have been reported previously
Although the first crossover sites were different from each other among the 10
The NAg3a amino acid of the Sabin 3 strain seems atypical; other type 3 wild poliovirus isolates that have circulated worldwide in recent years (after 1980) have sequences of NAg3a more like the Sabin 2 strain. But the type 3 wild poliovirus isolates from earlier year (before 1967) still have sequences of NAg3a more like the Sabin 3 strain. The possibility exist that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980. It seems likely, but is hard to prove, that perhaps the type 3/type 2 recombination events in the 3′ end of
The results of age estimation guided us towards the reconstruction of the potential histories of these clinical isolates. OPV strains had replicated in the human gut for 38–120 days (from the Ks estimate) and for 65–127 days (from the Kt estimate) after the initial OPV doses were given. Based on the fact that type 2 and type 3 vaccine viruses can replicate and be excreted by immunocompetent vaccinees for about 3 months after vaccination
The limitation with the method of the estimation of evolution used in this study is that this approach assumes a fixed substitution rate and multiplies it by the estimated number of nucleotide substitutions (total substitutions or synonymous substitutions), but in fact, the actual amount of evolution (number of substitutions) mainly depends on the virus effective population size inside the host and the frequency of replication. Additionally, the recombination events would lead to a change in the selection pressure, and most likely, there would be an increase in selection resulting in an increased evolutionary rate, meaning that the ages of the VP1 capsid recombinants may have been overestimated. This is most obvious in the case of CHN11144, whose estimated ages (65 days from the
All the children had one or more OPV before sampling, and the intervals between the dates of administration of last OPV and sampling are from 26 days to 1027 days, which indicated not all natural VP1 capsid recombinants directly derived from the OPV strains they received, most likely, some children were affected by them from environment. There was evidence that, like other vaccine-like polioviruses, VP1 capsid recombinants could survive and be detected from the environmental sewage
Genetic attenuation usually decreases viral fitness, and all the
Five of the VP1 capsid recombinants (CHN5275, CHN6060, CHN6218, CHN6356 and CHN12121) did not lose the temperature-sensitive phenotype of parent P3/Sabin strain. This result differed to three previously reported capsid recombinants which had almost entirely lost the temperature sensitivity
In conclusion, 10 natural type 3/type 2 intertypic
This study did not involve human participants and did not contain human experimentation, the only used material is stool samples collected from the AFP case patients for the purpose of public health initiated by World Health Organization and Chinese Ministry of Health, and the written informed consents from all participants involved in this study were obtained for the use of their stool samples. This study has been approved by the second session of Ethics Review Committee in Chinese Centre for Disease Control and Prevention.
10 type 3 polioviruses were isolated from nine AFP case patients (from Jiangxi, Henan, Yunnan, Guangdong, Gansu, Guizhou, Hubei, and Hebei provinces) and a healthy vaccinee (from Xizang Autonomous Region) during the period 2001–2008 in China. All the AFP case patients were less than 1.6 years old when they presented with symptoms, except for one patient from Hubei province (a 3.2-year-old boy). All of the children with AFP had at least one dose of OPV after birth; however, it was unknown when the healthy vaccinee from the Xizang Autonomous Region had received a dose of OPV because a written record of this could not be located. None of the patients with AFP showed signs of immunodeficiency at the time of presentation. Two stool specimens were collected from each of the nine AFP case patients at 24 hours apart within 14 days after onset of symptoms. One stool specimen was collected from the healthy vaccinee during an epidemiological survey of OPV coverage in 2007 (
RD (Human rhabdomyosarcoma) and L20B (mouse L cell expressing the human poliovirus receptor) cell lines were used to isolate viruses from the stool specimens by standard procedures
Viral RNAs were extracted from the viral isolates using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) and stored at −80°C for further use. 1µl (200U) SuperScript II ribonuclease H- reverse transcriptase (invitrogen, USA) was used to produce single stranded cDNA from 5µl of each purified viral RNA. The cDNA syntheses were primed by 7500A and Q8 (
Primer | Nucleotide position (nt) | Primer sequence (5-3) | Orientation | Reference |
0001S48 |
|
Forward |
|
|
EV/PCR-2 | 449–473 |
|
Forward |
|
EV/PCR-1 | 537–562 |
|
Reverse |
|
1275S | 1275–1294 |
|
Forward | This study |
1511A | 1492–1511 |
|
Reverse | This study |
1949S | 1949–1969 |
|
Forward | This study |
2224A | 2205–2224 |
|
Reverse | This study |
Y7 |
2397–2419 |
|
Forward |
|
2873A | 2853–2873 |
|
Reverse | This study |
3368S | 3368–3389 |
|
Forward | This study |
Q8 |
3477–3496 |
|
Reverse |
|
4443S | 4443–4465 |
|
Forward |
|
4489A | 4468–4489 |
|
Reverse |
|
5076S | 5076–5097 |
|
Forward | This study |
5274A | 5253–5274 |
|
Reverse | This study |
5904S | 5904–5929 |
|
Forward | This study |
6097A | 6077–6097 |
|
Reverse |
|
6914S | 6914–6934 |
|
Forward |
|
6970A | 6951–6970 |
|
Reverse | This study |
7500A |
|
Reverse |
|
The primer pairs Y7/7500A and 0010S48/Q8 are suggested for long distant PCR. The expected amplicons from these are 5.28 kb and 3.57 kb, respectively.
Two long-distant PCR amplifications (for one virus) were performed by using the TaqPlus Precision PCR system (Stratagene, USA), which consists of a blend of Stratagene cloned Pfu DNA polymerase (proof reading) and Taq2000 DNA polymerase (non-proof reading). Reactions contained 5µl of cDNA (see above), 0.1mM of each dNTP, 10µl of TaqPlus buffer, 1.0 ng/µl of a forward (0001S48 or Y7) and reverse (Q8 or 7500A) primer (
Two long-distant PCR products (for one virus) were purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). Cycle sequencing reactions were carried out using the version 3.0 of the BigDye terminator chemistry (Applied Biosystems), using the primers listed in
The sequences of the isolates were aligned with the reference strains by using the MEGA program v4.0 (Sudhir Kumar, Arizona State University, Arizona, USA)
Temperature sensitivities of 10 natural type 3/type 2 capsid-recombinant polioviruses were assayed on monolayer RD cells in 24-well plates as described before
The date of the initiating OPV dose for each patient was estimated from the
The complete genomic sequences of 10 natural type 3/type 2 capsid-recombinant polioviruses described in this study were deposited in the GenBank database under the accession numbers FJ859183 to FJ859192.
We thank Jianfeng Liu of Gansu Center for Disease Control and Prevention, Mei Hong of Xizang CDC, Huanying Zheng of Guangdong CDC, Xufang Ye of Guizhou CDC, Junmian Zhang of Hebei CDC, Xixiang Huo of Hubei CDC, Zhengrong Ding of Yunnan CDC, Ying Xiong of Jiangxi CDC, Sheng Zhao of Henan CDC, Liping Ren of Liaoning CDC, Miao Cheng of Beijing University of Chinese Medicine, for their excellent techniques and kind assistance with the laboratory investigation in this study.