Skip Navigation

Analysis of Reversions in the 5′-Untranslated Region of Attenuated Poliovirus after Sequential Administration of Inactivated and Oral Poliovirus Vaccines

  1. Majid Laassri1,
  2. Kathleen Lottenbach3,
  3. Robert Belshe3,
  4. Margaret Rennels2,
  5. Stanley Plotkin4 and
  6. Konstantin Chumakov1
  1. 1Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, and
  2. 2University of Maryland School of Medicine, Baltimore, Maryland;
  3. 3Saint Louis University, Saint Louis, Missouri;
  4. 4University of Pennsylvania and Sanofi Pasteur, Doylestown
  1. Reprints or correspondence: Dr. Konstantin Chumakov, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike HFM-470, Rockville, MD 20852 (chumakov{at}cber.fda.gov)
 
Next Section

Abstract

Replication of Sabin strains used in oral poliovirus vaccine (OPV) in the intestines of vaccine recipients leads to reversions that increase virus neurovirulence. Previously, a small study reported that prior immunization with inactivated poliovirus vaccine (IPV) resulted in faster accumulation of revertant virus, thus potentially increasing the risk of vaccine-associated paralytic poliomyelitis. We studied the impact that prior immunization with IPV and OPV has on shedding of revertant virus by healthy infants. By polymerase chain reaction (PCR), we amplified full-length poliovirus genomes directly from stool specimens from unimmunized infants and from infants previously immunized with IPV or OPV. The amplicons were used to quantify reversions in the 5′-untranslated region, using oligonucleotide microarray hybridization. Nearly all 140 samples that were PCR positive contained varying amounts of revertants of all 3 poliovirus serotypes. Polioviruses of Sabin types 2 and 3 reverted more easily than those of type 1. Prior vaccination with IPV did not increase the proportion of revertants after OPV administration

The worldwide use of live trivalent oral poliovirus vaccine (OPV) prepared from attenuated Sabin strains of poliovirus has resulted in eradication of poliomyelitis in the United States [1] and most other countries [2]. OPV has been widely used because of the ease of its administration, its low cost, and its ability to induce long-term systemic and local (mucosal) immunity [3]. However, this vaccine also causes rare but serious adverse reactions (vaccine-associated paralytic poliomyelitis [VAPP]) in primary vaccine recipients and their contacts [4]. Poliovirus strains isolated from patients with VAPP as well as from healthy vaccine recipients contain mutations that result in increased neurovirulence [5, 6]. Some of these mutations are direct reversions to the allele of wild-type progenitors of vaccine strains, whereas others are second-site suppressors of attenuated phenotype or are incidental changes. Among the best-characterized attenuating mutations in the Sabin strains are mutations located in the 5′-untranslated region (5′-UTR) [7]. These mutations have been identified in poliovirus of Sabin type 3 (472U→C) [5], type 2 (481A→G) [8], and type 1 (480G→A and 525U→C) [9] and are believed to selectively affect initiation of translation of viral polyprotein in neuronal cells [10, 11]. Isolation of the revertants from both patients with VAPP and healthy vaccinees [12, 13] suggests that host factors play a role in VAPP. However, the emergence of revertants is a necessary first step toward the reversion to full virulence that occurs after prolonged circulation of vaccine-derived polioviruses (cVDPVs). These fully virulent cVDPVs can cause small outbreaks of poliomyelitis, such as those documented in Egypt (1982–1993) [14], the Philippines (2001) [15], Hispaniola (2000–2001) [16], Madagascar (2002) [17], China (2004), and Laos (2005), illustrating a serious challenge to the development of prudent vaccination policies after global eradication is achieved. Therefore, studies of the factors affecting the reversion of attenuated poliovirus remain a high priority

To mitigate the shortcomings of both IPV and OPV, it has been proposed that the vaccines be used in combination—for instance, by giving 2 IPV doses followed by 2 OPV doses. In this case, IPV may provide immunity that would be sufficient to protect against VAPP, and subsequent administration of OPV would serve as a booster and result in strong intestinal immunity [18, 19]. Some countries adopted this immunization regimen, including the United States in the late 1990s before the Advisory Committee on Immunization Practices finally recommended the exclusive use of IPV [20, 21]. The results of one study conducted in the early 1990s suggested that prior immunization of children with IPV resulted in higher rates of reversion of attenuated poliovirus, raising potential safety concerns [18]. However, other studies produced contradictory information, and there was no convincing resolution of this issue [12]. The present study was designed to address this question

Recently, we published an extensive study of excretion of vaccine poliovirus by healthy vaccine recipients after administration of OPV, IPV, or a combination thereof [22]. To detect and quantify virus, we used a new approach based on polymerase chain reaction (PCR) amplification of viral full-length cDNA (FL-PCR) directly from stool samples from vaccine recipients [19]. The direct amplification protocol allowed us to circumvent the use of cell cultures to produce virus from clinical samples and provided a rapid method of isolating viral genomes. It also ensured a more accurate quantification of mutations occurring after the administration of OPV, because growth in cell cultures might alter the mutational composition of the viral population. We found that the sensitivity of the assay was comparable to that of cell culture isolation protocols. In the present study, we also used this direct FL-PCR protocol to amplify poliovirus cDNA from a large number of stool samples collected after administration of different regimens of OPV and IPV. We then performed an analysis of reversion in the 5′-UTR of the 3 serotypes of OPV by use of hybridization with newly developed microarrays of immobilized oligonucleotides (5′-UTR microchips). The microchips contain short oligonucleotides immobilized on the glass surface and can distinguish between attenuated and revertant nucleotides present in viral cDNA. This microchip-based method for mutant quantification allowed us to process a large number of samples and to obtain quantitative information about the contents of revertants in each of the 3 serotypes of poliovirus separately

Previous SectionNext Section

Subjects, Materials, and Methods

Subjects, vaccines, and specimen collectionHealthy infants were enrolled in a study aimed at the comparative evaluation of combined vaccination regimens performed in 1997 at Saint Louis University and at the University of Maryland [23]. Subgroups of these subjects were evaluated for virus shedding and virulent poliovirus reversion rates in OPV/OPV versus IPV/OPV schedules. The study was approved by the institutional review boards of the respective institutions, and informed consent was obtained from parents of participating subjects

In the primary study, 527 infants were randomized equally into 4 arms. Subjects in arms A and B received the trivalent OPV Orimune (Wyeth-Lederle Vaccines and Pediatrics) at 2, 4, and 6 months of age. Subjects in arm C received enhanced IPV (IPOL; Connaught-Pasteur Merieux) at 2 and 4 months of age, followed by OPV at 6 months of age. Subjects in arm D received IPV at 2, 4, and 6 months of age. A total of 259 stool specimens were obtained from study subjects in arms A, B, and C at 1 week and 3 weeks after doses 1 and 3. These were used in previous work to evaluate OPV shedding [22], and the 140 samples that were found to be positive by FL-PCR were further evaluated in this study, to identify and quantify the 5′-UTR reversions in all 3 poliovirus serotypes. For subjects in the OPV/OPV/OPV group (arms A and B), 44 specimens were obtained at 1 week and 39 were obtained at 3 weeks after dose 1 (subgroup 1), and 9 FL-PCR–positive specimens were obtained at 1 week and 2 were obtained at 3 weeks after dose 3 (subgroup 2). For subjects in the IPV/IPV/OPV group, 32 FL-PCR–positive specimens were obtained at 1 week and 14 were obtained at 3 weeks after dose 3 (subgroup 3). These samples were analyzed for the presence of cDNA of each poliovirus serotype (table 1) and were used in this study for detection and quantification of known reversions in the 5′-UTR

Figure 1
View larger version:
Figure 1

Hybridization of fluorescently labeled poliovirus cDNA with microarrays of immobilized oligonucleotides. Each row contained 10 replicates of oligoprobes specific to point mutations in the 5′-untranslated region of poliovirus. Two mutations (480A and 525C) were quantified in Sabin type 1 derivative strains, and 1 mutation each was quantified in Sabin type 2 and Sabin type 3 (481G and 472C, respectively)

Figure 2
View larger version:
Figure 2

Contents of neurovirulent mutations in the 5′-untranslated region of attenuated polioviruses in stool samples from infants immunized with oral poliovirus (OPV), inactivated poliovirus (IPV), and a combination thereof. Black circles denote samples from individual subjects, and white squares denote averages for each group

Table 1
View larger version:
Table 1

Excretion of poliovirus, by individual serotype

VirusesSabin strains of types 1, 2, and 3 (GenBank accession numbers AY184219, AY184220, and AY184221, respectively) were US neurovirulence reference samples. Stocks of vaccine-derived poliovirus isolate 11262 (“99/056-252-14”; GenBank accession number AF462418) and of wild-type Mahoney and Leon/37 strains (GenBank accession numbers NC_002058 and K01392, respectively), as well as vaccine-derived poliovirus isolate 154, were grown in HEp-2 cells. Viral RNA was extracted from clarified cell culture fluid, converted to cDNA by use of SuperScript II reverse transcriptase and primers complementary to the 3′ end of poliovirus RNA, and amplified by FL-PCR, as described below for stool samples

RNA extraction, cDNA preparation, and amplification of full-length OPV genome in stool specimensThe viral RNA extraction, cDNA preparation, and amplification of full-length genome were performed directly from stool specimens, as described elsewhere [19]. Briefly, 1 g of frozen stool was vortexed in 10 mL of Dulbecco’s PBS and centrifuged for 10 min at 325 g and supernatants were aliquoted (1.5 mL/vial) and stored frozen at −70°C. A total of 140 μL of the stool supernatant was added to a QIAamp Viral RNA Mini Kit (QIAGEN) for isolation of RNA, in accordance with the manufacturer’s protocol. The extracted RNA was eluted in a final volume of 60 μL of sterile RNase-free water

For viral cDNA preparation, 10 μL of RNA was added to a reaction that contained 1 mmol/L dithiothreitol, 2.5 μg/mL concentrations of each primer (A7-sabin1,3 and A7-sabin2), 0.5 mmol/L dNTP mix, and 1× first-strand RT buffer (Life Technologies). The final volume of the reaction mix was 50 μL. The mixture was heated for 5 min at 65°C and then quickly chilled on ice. Superscript II (12 U/μL) was added to the mixture and incubated for 2 h at 42°C, then additional Superscript II (4 U/μL) was added, and the mixture was incubated for another 3 h at 42°C

Direct amplification of full-length poliovirus genome from stool specimens was performed by PCR as described previously [19]; 10 μL of cDNA was used for full-length PCR amplification of the poliovirus genome. The reaction was performed in accordance with the manufacturer’s manual for the XL-PCR kit (Perkin-Elmer/ABI), using a GeneAmp 9700 thermocycler (ABI). The following temperature conditions were used: preincubation for 30 s at 94°C, followed by 35 cycles each consisting of 15 s at 94°C and 10 min at 65°C, and then run-off incubation for 30 min at 72°C

Oligonucleotide microarray hybridization analysis5′-UTR microchips were created for the identification and quantification of the 4 known reversions in the 5′-UTR of the 3 poliovirus strains (nt 480 and 525 for Sabin type 1, nt 481 for Sabin type 2, and nt 472 for Sabin type 3) and have been described in detail elsewhere [19]. Briefly, the 5′-UTR microchips contained 10 spots of oligoprobe for a specific Sabin strain and 10 spots of oligoprobe specific for revertant virus. Microarrays also contained 2 oligonucleotides specific for a conserved region (control). Each control was spotted 5 times in the last row. The redundant spotting was used to increase quantification accuracy. Ten individual microarrays for 5′-UTR analysis were spotted on each slide. Hybridization probes were single-stranded DNA prepared by asymmetric PCR, using a Perkin Elmer PCR kit as described elsewhere [19]. The single-stranded DNA was purified by use of a QIAquick PCR purification kit (QIAGEN) and was diluted in 50 μL of water. Aliquots containing 0.2 μmol/L single-stranded DNA were labeled with a Cy5 or Cy3 Micromax ASAP RNA Labeling Kit (Perkin-Elmer) and purified using CENTRI-SEP Spin columns (Princeton Separation)

Several microarrays spotted on the same slide were simultaneously hybridized for at least 30 min at 45°C with fluorescently labeled DNA samples prepared from the reference Sabin strain, reference Sabin revertant (or wild-type poliovirus), and 1 or more test strains. The microarray was then washed for 2 min in 2× standard saline citrate (SSC) with 0.1% SDS, followed by 1 min in 2× SSC

Microarray images were taken using a confocal fluorescent scanner ScanArray 5000 (GSI Lumonics) equipped with green and red HeNe lasers (543 nm and 632 nm, for excitation of Cy3 and Cy5, respectively). Images were then analyzed using QuantArray software (Packard BioScience). The values obtained from 5′-UTR microchips were normalized, and the percentage of reversion was calculated by dividing the normalized signal from each revertant oligoprobe by the total signal (signal obtained from both revertant and vaccine oligoprobes). The numbers obtained from 10 replicates of each oligoprobe (vaccine, revertant) were averaged, and the SD was calculated

Previous SectionNext Section

Results and Discussion

Earlier studies have demonstrated that, in each strain of Sabin poliovirus, mutations that lead to higher neurovirulence occur during replication in vitro and in vivo. It has also been reported that the proportion of revertants in vaccine recipients may depend on prior immunization status [18]. Therefore, we conducted this study of healthy infants immunized with 2 doses of OPV or 2 doses of IPV and then challenged with an additional dose of OPV, to study virus excretion and to determine the contents of neurovirulent reversions. To detect and quantify the reversions, we used a previously developed method [19] to prepare viral cDNA directly from stool samples and to quantify the reversions in the 5′-UTR by the oligonucleotide microarray (5′-UTR microchip) method

Of 259 stool samples obtained from children immunized with different combinations of OPV and IPV, 140 were found to be positive by FL-PCR for the presence of poliovirus (table 1). Children who were previously immunized with OPV or IPV developed mucosal immunity and excreted poliovirus at a lower frequency, compared with children who received the first dose of OPV

The PCR-amplified viral cDNA prepared directly from the stool specimens was then used for quantification of reversions in the 5′-UTR separately for each of the 3 poliovirus serotypes. Fluorescently labeled DNA for hybridization was prepared by PCR amplification using primers specific for each poliovirus serotype. Figure 1 shows hybridization patterns obtained for vaccine and revertant (wild-type) reference strains of all 3 serotypes, as well as 1 example for cDNA derived from a stool sample from a vaccine recipient shedding a mixture of attenuated and revertant viruses. Reversions at 1 nt were determined for Sabin type 2 and Sabin type 3 viruses (481G and 472C), whereas, for the Sabin type 1 strain, we quantified both direct reversion to the wild-type genotype (480A) and compensatory mutation in the complementary nucleotide (525C). The intensity of microarray hybridization with vaccine-specific and revertant-specific oligoprobes was determined by confocal laser scanning of the fluorescent image and used to calculate the contents of revertants in a sample. Figure 2 summarizes the quantitative findings for all PCR-positive samples and shows that the majority of samples from children who received OPV with or without prior IPV contained revertants. The average proportions of revertants were higher in 3-week samples than in 1-week samples, suggesting that the accumulation of mutations is a gradual process (table 2). The proportion of revertants also differed for individual serotypes of poliovirus and was highest for Sabin type 2 and lowest for Sabin type 1. The data on the genetic stability of attenuated poliovirus in vivo presented in this article are in general agreement with the conclusions made in early studies from Minor’s laboratory [24, 25], except that we found that 5′-UTR mutations in the Sabin type 2 component of OPV—and not Sabin 3, as previously thought—were the least stable

Table 2
View larger version:
Table 2

Average contents of revertants in stool from oral poliovirus vaccine (OPV) recipients after different regimens of inactivated poliovirus vaccine (IPV) and OPV

Comparison of reversion frequencies in vaccinees after different vaccine schedules showed that there was no correlation with whether subjects were receiving their first dose of OPV or had been previously immunized with either OPV or IPV (figure 2). The number of positive samples in the group of children who received 3 doses of OPV was very low, making it difficult to accurately determine the proportion of revertants; however, on the basis of the available data, it appears that prior immunization with 2 doses of OPV did not affect the proportion of revertants. Immunization with 2 doses of IPV also did not affect the proportion of revertants, contrary to previous data published by Ogra et al. [18] suggesting that prior administration of IPV does not carry an additional risk of inducing VAPP or creating cVDPVs

Although the global campaign to eradicate poliomyelitis is lagging behind schedule and the target date is continuously being revised, there is still hope that it will eventually succeed and that the circulation of wild-type poliovirus will be stopped worldwide. In that case, paralysis caused by vaccine derivatives will be the only “naturally” occurring poliomyelitis that remains. One radical solution to this problem that is currently advocated by the World Health Organization is to stop the use of OPV altogether after the eradication of wild-type virus. This proposal remains controversial, because it will leave huge segments of the human population unprotected from accidental or malicious reintroduction of poliovirus back into circulation; therefore, the OPV cessation scenario involves serious ethical, legal, and political issues. The world is obviously facing the dilemma of whether to continue immunization with OPV and thereby induce paralysis in hundreds of vaccine recipients per year worldwide or to withhold protection from the majority of the world’s population and create the risk of a potentially catastrophic outbreak of poliomyelitis. A prudent solution would be to replace OPV with the safer IPV, but such a switch will require the creation of production facilities to make IPV for the entire world population, which would require substantial resources. Therefore, the reduction of adverse reactions caused by OPV may provide at least a temporary solution to the dilemma, and the IPV/OPV combination, which would reduce the number of needed IPV doses by half, could be one of the options. Use of 1 or 2 IPV doses before administering OPV would reduce the demand for the more expensive IPV and should provide sufficient protection against VAPP. Experience in the United States and other developed countries suggests that this strategy does work. Clinical trials in countries currently using OPV may provide an answer to the question of whether the same strategy would work in other settings

Previous SectionNext Section

Acknowledgment

We thank Eugenia Dragunsky for her suggestions and critical review of this article

Previous SectionNext Section

Footnotes

  • ↵(See the editorial commentary by Kew, on pages 1341–3.)

  • ↵Potential conflicts of interest: S.P. is the executive adviser to the CEO of Sanofi Pasteur, and M.R. is the chairperson of the multicenter vaccine safety trial monitoring board for Sanofi Pasteur. All other authors report no conflicts of interest or commercial associations

    Financial support: Defense Advanced Research Projects Agency (grant to K.C.); National Vaccine Program Office (grant to K.C.); National Institute of Allergy and Infectious Diseases (contracts NO1-AI-45250 and NO1-AI-45251 with R.B. and M.R.)

  • Received August 5, 2005.
  • Accepted October 28, 2005.
Previous Section
 

References

  1. ↵
    1. Strebel PM,
    2. Sutter RW,
    3. Cochi SL,
    4. et al
    . Epidemiology of poliomyelitis in the United States one decade after the last reported case of indigenous wild virus-associated disease. Clin Infect Dis 1992;14:568-79.
    Abstract/FREE Full Text
  2. ↵
    1. Centers for Disease Control and Prevention
    . Certification of poliomyelitis eradication—European Region, June 2002. MMWR Morb Mortal Wkly Rep 2002;51:572-4.
    Medline
  3. ↵
    1. Ghendon Y,
    2. Robertson SE
    . Interrupting the transmission of wild polioviruses with vaccines: immunological considerations. Bull World Health Organ 1994;72:973-83.
    MedlineWeb of Science
  4. ↵
    1. Prevots DR,
    2. Strebel PM
    . Poliomyelitis prevention in the United States: new recommendations for routine childhood vaccination place greater reliance on inactivated poliovirus vaccine. Pediatr Ann 1997;26:378-83.
    MedlineWeb of Science
  5. ↵
    1. Cann AJ,
    2. Stanway G,
    3. Hughes PJ,
    4. et al
    . Reversion to neurovirulence of the live-attenuated Sabin type 3 oral poliovirus vaccine. Nucleic Acids Res 1984;12:7787-92.
    Abstract/FREE Full Text
  6. ↵
    1. Friedrich F
    . Genomic modifications in Sabin vaccine strains isolated from vaccination-associated cases, healthy contacts and healthy vaccinees. Acta Virol 1996;40:157-70.
    MedlineWeb of Science
  7. ↵
    1. Minor PD
    . The molecular biology of poliovaccines. J Gen Virol 1992;73:3065-77.
    Abstract/FREE Full Text
  8. ↵
    1. Macadam AJ,
    2. Pollard SR,
    3. Ferguson G,
    4. et al
    . Genetic basis of attenuation of the Sabin type 2 vaccine strain of poliovirus in primates. Virology 1993;192:18-26.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Otelea D,
    2. Guillot S,
    3. Furione M,
    4. et al
    . Genomic modifications in naturally occurring neurovirulent revertants of Sabin 1 polioviruses. Dev Biol Stand 1993;78:33-8.
    MedlineWeb of Science
  10. ↵
    1. Svitkin YV,
    2. Cammack N,
    3. Minor PD,
    4. Almond JW
    . Translation deficiency of the Sabin type 3 poliovirus genome: association with an attenuating mutation C472----U. Virology 1990;175:103-9.
    CrossRefMedlineWeb of Science
  11. ↵
    1. Guest S,
    2. Pilipenko E,
    3. Sharma K,
    4. Chumakov K,
    5. Roos RP
    . Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3. J Virol 2004;78:11097-107.
    Abstract/FREE Full Text
  12. ↵
    1. Abraham R,
    2. Minor P,
    3. Dunn G,
    4. Modlin JF,
    5. Ogra PL
    . Shedding of virulent poliovirus revertants during immunization with oral poliovirus vaccine after prior immunization with inactivated polio vaccine. J Infect Dis 1993;168:1105-9.
    Abstract/FREE Full Text
  13. ↵
    1. Dunn G,
    2. Begg NT,
    3. Cammack N,
    4. Minor PD
    . Virus excretion and mutation by infants following primary vaccination with live oral poliovaccine from two sources. J Med Virol 1990;32:92-5.
    MedlineWeb of Science
  14. ↵
    1. Centers for Disease Control and Prevention
    . Circulation of a type 2 vaccine-derived poliovirus— Egypt, 1982–1993. MMWR Morb Mortal Wkly Rep 2001;50:41-42. 51.
    Medline
  15. ↵
    1. Centers for Disease Control and Prevention
    . Acute flaccid paralysis associated with circulating vaccine-derived poliovirus, Philippines, 2001. Wkly Epidemiol Rec 2001;76:319-20.
    Medline
  16. ↵
    1. Kew O,
    2. Morris-Glasgow V,
    3. Landaverde M,
    4. et al
    . Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus. Science 2002;296:356-9.
    Abstract/FREE Full Text
  17. ↵
    1. Centers for Disease Control and Prevention
    . Paralytic poliomyelitis in Madagascar, 2002. Wkly Epidemiol Rec 2002;77:241-2.
    Medline
  18. ↵
    1. Ogra PL,
    2. Faden HS,
    3. Abraham R,
    4. Duffy LC,
    5. Sun M
    . Effect of prior immunity on the shedding of virulent revertant virus in feces after oral immunization with live attenuated poliovirus vaccines. J Infect Dis 1991;164:191-4.
    Abstract/FREE Full Text
  19. ↵
    1. Laassri M,
    2. Dragunsky E,
    3. Enterline J,
    4. et al
    . Genomic analysis of vaccine-derived poliovirus strains in stool specimens by combination of full-length PCR and oligonucleotide microarray hybridization. J Clin Microbiol 2005;43:2886-94.
    Abstract/FREE Full Text
  20. ↵
    1. Poliomyelitis prevention: recommendations for use of inactivated poliovirus vaccine and live oral poliovirus vaccine
    . American Academy of Pediatrics Committee on Infectious Diseases. Pediatrics 1997;99:300-5.
    Abstract/FREE Full Text
  21. ↵
    1. Advisory Committee on Immunization Practices
    . Poliomyelitis prevention in the United States: introduction of a sequential vaccination schedule of inactivated poliovirus vaccine followed by oral poliovirus vaccine; recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 1997;46(RR-3):1-25.
    Medline
  22. ↵
    1. Laassri M,
    2. Lottenbach K,
    3. Belshe R,
    4. et al
    . Effect of different vaccination schedules on excretion of oral poliovirus vaccine strains. J Infect Dis 2005;192:2092-8.
    Abstract/FREE Full Text
  23. ↵
    1. Rennels MB,
    2. Englund JA,
    3. Bernstein DI,
    4. et al
    . Diminution of the anti-polyribosylribitol phosphate response to a combined diphtheria-tetanus-acellular pertussis/Haemophilus influenzae type b vaccine by concurrent inactivated poliovirus vaccination. Pediatr Infect Dis J 2000;19:417-23.
    CrossRefMedlineWeb of Science
  24. ↵
    1. Minor PD,
    2. Dunn G
    . The effect of sequences in the 5′ non-coding region on the replication of polioviruses in the human gut. J Gen Virol 1988;69:1091-6.
    Abstract/FREE Full Text
  25. ↵
    1. Minor PD,
    2. Dunn G,
    3. Ramsay ME,
    4. Brown D
    . Effect of different immunization schedules on the excretion and reversion of oral polio vaccine strains. J Med Virol 2005;75:153-60.
    CrossRefMedlineWeb of Science
| Table of Contents

This Article

  1. J Infect Dis. (2006) 193 (10): 1344-1349. doi: 10.1086/503366
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most