MAJOR ARTICLE
Shedding of Sabin Poliovirus Type 3 Containing
the Nucleotide 472 Uracil-to-Cytosine Point Mutation
after Administration of Oral Poliovirus Vaccine
Claudia V. Martinez,a Matt O. Old,a Douglas K. Kwock,a Shalla S. Khan,a Joaquin J. Garcia,a Christina S. Chan,a
Ramothea Webster,a Meira S. Falkovitz-Halpern, and Yvonne A. Maldonado
Department of Pediatrics, Stanford University School of Medicine, Stanford, California
A uracil-to-cytosine point mutation at nucleotide (nt) 472 of Sabin oral poliovirus vaccine (OPV) type 3 is
found in conjunction with vaccine-associated paralytic poliomyelitis (VAPP). Direct RNA extraction and mutant
analysis by polymerase chain reaction and restriction enzyme cleavage were used to identify this point mutation
in clinical samples. A total of 238 stool samples were obtained from 28 healthy infants for 6 weeks after OPV
vaccination. More than 25% of infants shed OPV3 in the week after vaccination, with a decrease on day 6. A
second wave of OPV3 shedding occurred beginning the second week after vaccination and was maintained
through the end of the study period. During the first week after vaccination, the proportion of nt 472 mutants
in the shed OPV3 increased from undetectable to almost 100%. During the second shedding period, the
proportion of nt 472 mutants remained close to 100%. These results suggest that selective mutation drives
the VAPP-associated nt 472 point mutation for OPV3 in the human gastrointestinal tract.
Highly attenuated polioviruses, representing each of the
3 human serotypes, have been used as vaccine viruses
for 140 years [1–3]. Sabin oral poliovirus vaccine
(OPV) consists of live attenuated forms of 3 poliovirus
serotypes—OPV1, OPV2, and OPV3. OPV has been
widely used because of its ease of oral administration,
low production costs for developing countries, ability
to induce both humoral and mucosal immunity, and
rapid induction of long-term immunity [4]. Despite
Received 30 May 2003; accepted 12 January 2004; electronically published 18
June 2004.
Presented in part: American Pediatric Society, New Orleans, April 1998; American
Pediatric Society and The Society for Pediatric Research, San Francisco, May 1999.
Financial support: Child Health Research Fund, Lucile Salter Packard Children’s
Hospital at Stanford University Medical Center.
a
Present affiliations: University of Rochester School of Medicine and Dentistry,
Rochester, New York (C.V.M. and R.W.); University of Texas Houston School of
Medicine, Houston (M.O.O.); University of Hawaii, John A. Burns School of Medicine,
and Kapiolani Medical Center for Women and Children, Honolulu (D.K.K.); Touro
University, College of Osteopathic Medicine, San Jose, California (S.S.K.); Stanford
University School of Medicine, Stanford, California (J.J.G.); University of California
School of Medicine, San Diego (C.S.C.).
Reprints or correspondence: Dr. Yvonne A. Maldonado, Dept. of Pediatrics, Stanford
University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305 (bonniem@
stanford.edu).
The Journal of Infectious Diseases 2004; 190:409–16
2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/19002-0028$15.00
these benefits, OPV has been discontinued in the United
States because of the small but preventable risk of vaccine-associated paralytic poliomyelitis (VAPP) associated
with OPV. The Advisory Committee on Immunization
Practices of the Centers for Disease Control and Prevention and other organizations currently recommend
vaccination with a parenterally administered, enhancedpotency inactivated poliovirus vaccine (e-IPV) series at
2, 4, and 6–18 months and 4–6 years of age [5–7].
It is well documented that Sabin serotype–specific
point mutations exist in a large proportion of cases of
VAPP [8–14]. Of the 3 human poliovirus serotypes,
OPV3 is the one most often associated with VAPP [15].
Despite the high rate of OPV3 VAPP–associated point
mutations in humans, as identified by conventional tissue-culture methods, the risk of VAPP is extremely low
(∼1 case/2.4 million doses of OPV administered [16]).
The rapid development of humoral immunity to OPV
after administration and the low virulence of attenuated
vaccine polioviruses likely are responsible for the low
incidence of VAPP, as demonstrated by the high incidence of VAPP among patients with selective humoral
immunodeficiencies [15, 17–19]. However, the low incidence of VAPP may also occur, in part, because the
Detection of Sabin Poliovirus Mutants by RNA Extraction and MAPREC • JID 2004:190 (15 July) • 409
accumulation of in vivo VAPP-associated point mutations is
amplified in vitro by tissue-cultivation methods traditionally
used to recover viruses from clinical samples. As is true of all
viruses, OPV serotypes undergo rapid genomic modifications
during in vitro cell passage and after administration to humans
and animals [20–23]. These modifications may occur as recombination or mutation events and, at some sites, appear to
be recurring, specific genetic changes, as documented by the
existence of VAPP-associated point mutations. Whether these
modifications are due to an actual difference in the frequency
of mutation events or to the unequal viability of the modified
viral genomes is not known. However, the observation that
replication of poliovirus in the human intestinal tract and in
cell lines results in recurring mutation patterns suggests that
unique selection pressures exist in vivo and in vitro. Direct
extraction of Sabin vaccine virus RNA from clinical samples
could provide not only a rapid method of isolating viral genomes but also a more accurate measure of in vivo mutations
occurring after administration of OPV.
In a previous study, we described a modified guanidine thiocyanate (GuSCN) extraction method used to isolate OPV3 RNA
directly from clinical samples [24]. This nucleic acid precipitation–free technique allowed for the circumvention of intermediate cell lines or animal models in extracting viral RNA
from stool samples and for the isolation of populations of viral
genome fragments from individual clinical samples. We demonstrated that this method identified Sabin type 3 isolates from
70% of tissue culture–positive and 10% of tissue culture–negative samples and that the sensitivity of the assay reached a
dilution of 103 TCID50/0.1 mL. In the present study, we used
this GuSCN viral RNA–isolation method to collect large numbers of unmodified viral genomes from humans after administration of OPV. We then sought to conduct in vivo analysis
of the proportion of mutant OPV3 genomes by use of mutant
analysis by polymerase chain reaction (PCR) and restriction
enzyme cleavage (MAPREC). MAPREC is a molecular assay
developed at the Food and Drug Administration (FDA) for
assessing the quality of OPV vaccine lots before their release
for commercial use [25, 26]. It was developed as an alternative
to the reference standard, the monkey neurovirulence test
(MNVT), which is cumbersome, costly, and time-consuming.
MAPREC uses PCR and site-specific enzyme cleavage of selected genome sequences to estimate the proportion of point
mutations associated with neurovirulence in OPV1, OPV2, and
OPV3 vaccine isolates. In studies at the FDA, MAPREC identified all OPV3 batches that failed the MNVT [27]. These OPV3
batches were shown to contain ⭓0.9% OPV3 genomes expressing the neurorevertant UrC point mutation at nt 472.
In the present study, we used our direct-extraction method
with a modified MAPREC procedure to describe temporal
shedding patterns and development of the VAPP-associated
410 • JID 2004:190 (15 July) • Martinez et al.
UrC point mutation at nt 472 in OPV3 genomes after administration of OPV. The combination of RNA extraction and
MAPREC allows analysis of poliovirus population-based replication and mutation dynamics in vivo.
PATIENTS, MATERIALS, AND METHODS
Study population. The human-experimentation guidelines of
the US Department of Health and Human Services and the
Committee for the Protection of Human Subjects at Stanford
University were followed in the conduct of clinical research. The
study was conducted over the course of 10 months, from February 1998 to December 1998. Twenty-eight healthy 1-year-old
infants were recruited from the Ambulatory Care Clinic at the
Lucile Salter Packard Children’s Hospital (LPCH; Stanford, CA).
All healthy full-term infants who completed primary poliovirus
vaccination (2 doses of e-IPV at 4 and 6 months of age) within
the prior year and who attended the clinic for their third poliovirus vaccination with OPV were eligible for enrollment. Parents
of potential study patients were contacted by telephone before
visiting the LPCH clinic, informed consent was obtained, and
parents were asked to bring a baseline stool sample to the clinic
on the day of OPV vaccination. Parents were then asked to obtain
1 stool sample/day for 1 week after vaccination and 1 sample at
the end of the second, third, fourth, and sixth weeks (days 14,
21, 28, and 42) after OPV vaccination. Stool samples were stored
in an airtight container provided to the families, which was collected from their homes once a week. Stool samples were packed
into labeled 2.0-mL microcentrifuge tubes (Applied Scientific)
and were stored at ⫺80C until further testing for the presence
of OPV3 genome by GuSCN extraction.
Preparation of OPV3-specific primers. Primers created by
Genset were used to amplify a conserved sequence in OPV3
strain Sabin-Leon a1b. All reverse-transcription (RT) PCRs
used the antisense primer (designated “bS3–721”) from the 5
noncoding region of the OPV3 genome (figure 1). Samples and
constructs were identified with the sense primer (designated
“S3+432”). These OPV3-specific primers amplify a 290-bp region, which is located in a highly conserved region of the OPV3
genome. Since the area of interest does not contain a common
restriction site, the sense primer was prepared with a base pair
mismatch, to incorporate a restriction site for EcoRI. The sense
primer was mismatched at nt 468, so that an EcoRI site was
created on nonmutant nt 472 U OPV3 fragments, but not on
fragments containing the nt 472 C mutation. The restriction
site was designed to appear only when the nonmutant sequence
not containing the point mutation at nt 472 was amplified.
Preparation and application of controls. Control constructs
were prepared using bS3–721 and specific sense primers (designated “S3+432/472T” and “S3+432/472C”) (figure 1). S3+432/
472T was used for preparation of the nonmutant construct, and
S3+432/472C was used for preparation of the mutant construct.
Figure 1. Oral poliovirus vaccine (OPV) type 3 primers and amplicon sequence. A, 1: Sabin type 3–specific primer pairs, designated “bS3⫺721” and
“S3+432,” and polymerase chain reaction (PCR) product sequences; 1.1: Antisense (bS3⫺721) and sense (S3+432) primers. Antisense primer is labeled
with biotin (b) for a nonrelated application; 1.2: Sense primer is mismatched at 468 to incorporate an EcoRI site if the nonmutant is present. A, 2: Sabin
type 3–specific primer pairs for construct preparation. Both constructs were prepared with the antisense primer bS3⫺721; 2.1: Sense primer (S3+432/
472T) for nonmutant construct preparation; 2.2: Sense primer (S3+432/472C) for mutant construct preparation. “G” denotes the incorporated nucleotide
base-pair mismatch for nonmutant nt 472 U OPV3 fragments. B, Sabin type 3–specific sequences amplified by reverse-transcription polymerase chain
reaction. Position 472 is variable. “T” denotes the nonmutant and is subjected to EcoRI restriction endonuclease. “C” denotes the mutant and is resistant
to EcoRI restriction endonuclease.
OPV3 and Sabin vaccine poliovirus type 3 P3 Leon stock were
provided by Dr. David Schnurr (Department of Health Services,
Berkeley, CA). OPV3 (Sabin-Leon 12a1b) was used as the template for the nonmutant construct, and P3 Leon (Leon/37) was
used as the template for the mutant construct. Published hybridization methods were used to create pure DNA constructs
of the mutant and nonmutant viral sequences [28].
Preparation of samples. Direct extraction of poliovirus
RNA was accomplished by use of a modified GuSCN extraction
method originally designed for the extraction of other enteric
viral RNA from stool samples [24, 29–33]. After extraction, the
purified RNA was immediately placed on ice, if RT-PCR was
to be performed, or was frozen at ⫺20C for later testing.
RT-PCR of 290-bp region with avian myoblastosis virus
reverse-transcriptase. After extraction of viral RNA from
stool samples, RT-PCR was performed by use of a programmable thermal cycler (PerkinElmer Cetus), according to meth-
ods published elsewhere [24]. After RT-PCR, the samples were
either processed immediately for visualization on an electrophoresis gel, to detect the presence of amplified 290-bp sequences, or were stored at ⫺20C for later testing. The samples
were run on 3% agarose gels, stained with ethidium bromide,
and visualized by use of the Gel Doc 1000 Macintosh UV
Fluorescent Documentation System (BioRad).
Identification and quantification of mutant genome.
MAPREC preparations were made from each positive sample.
Two sterile 0.5-mL reaction tubes containing 8.5 mL of the
positive sample, mixed with 1 mL of SuRE/Cut Buffer H (Roche
Applied Scientific), were prepared. EcoR1 (0.5 mL), at a concentration of 40 active units/mL (Boehringer Mannheim), was
added to 1 of the reaction tubes to make the digestion tube,
and 0.5 mL of dH2O was added to the other to make the undigested control. The reaction tubes were incubated for 1 h at
37C and for 5 min at 65C. After the incubation, 1.1 mL of
Detection of Sabin Poliovirus Mutants by RNA Extraction and MAPREC • JID 2004:190 (15 July) • 411
10⫻ loading dye (50% glycerol, 50% Tris-acetate/EDTA [TAE]
electrophoresis buffer, and 0.25% bromophenol blue; Sigma
Chemical) was added to each reaction tube. The reaction products were run on a 3% TAE agarose gel for 3–5 h in a BioRad
Sub Cell GT gel box (BioRad) at 100 V. Constructs of mutant
and nonmutant sequences were run in parallel to the reaction
products, as controls for fragment digestion. Ladders of 100
and 25 bp (Gibco BRL Life Technologies) were run in parallel
for scaling. A gel documentation system (Gel Doc 1000-Macintosh UV Fluorescent Documentation System; BioRad) was
used to create a picture of the gels.
The UV fluorescence of the bands representing 290 and 253
bp, in the digested and undigested lanes of each extract, were
read from the picture of the gel by 1–3 people, by use of
Quantity One (version 4; BioRad) and Molecular Analyst (version 2.1.1; BioRad). All the gels read by only 1 person had very
low signal-to-background ratios and were discarded as having
poor quality; thus, all the gels used for calculating mutant proportions (MPs) were read by 2–3 people. The ratio of signal
to background was estimated for all the lanes, as well as the
intensity of PCR artifact in the 253-bp region relative to the
true signal in the 290-bp regions of the undigested lanes of the
extracts (shown in Appendix A, which appears only in the
electronic edition of the Journal). These estimates were used
to determine the level of reliability (LR)—high, medium, or
low—for each analysis of an MAPREC preparation. Two to five
MAPREC preparation sets were made from each positive sample. The MP of each preparation was determined from the
intensity of the bands in both the digested and the undigested
lanes (shown in Appendix B, which appears only in the electronic edition of the Journal). The MP of a given sample at
each LR was obtained by averaging the estimates obtained from
those analyses of the preparations that satisfied the conditions
of that LR or higher. The resulting MP of the MAPREC preparations made from a given sample were averaged to produce
the estimated MP of the sample. The data were analyzed by
use of SAS (version 8.01; SAS Institute).
Nucleotide sequence data. The sequence for OPV3 was
found and analyzed on GenBank for the conserved sequences
containing identifiable point mutations that could be easily replicated using this procedure. The accession number for OPV3
strain Sabin-Leon 12a1b is X00925-K00043 [34].
RESULTS
Shedding of OPV3. Stool samples (n p 238 ) were obtained
from the 28 infants enrolled in the present study. All 12 samples
obtained before the administration of OPV were negative for
OPV3. Of the remaining 226 samples, 44 (19%) were positive
for OPV3 by the GuSCN extraction method. Figure 2 shows
the biphasic fecal shedding pattern of OPV3. Peak shedding
occurred on day 3, with 35% of the infants (8/23) shedding
412 • JID 2004:190 (15 July) • Martinez et al.
Figure 2. Daily no. and proportion of oral poliovirus vaccine (OPV) type
3–positive stool samples from the 28 study infants obtained after administration of OPV.
OPV3. Shedding decreased to 1 (4%) of 23 infants on day 6
and 1 (6%) of 18 infants on day 7. This decrease is statistically
significant (P p .06 if daily counts are considered; P p .005 if
days 2 and 3, 4 and 5, and 6 and 7 are grouped, Fisher’s exact
test). During the second week, OPV3 was detected in ⭓20%
of the samples and remained at that level for the 6-week samplecollection period (P p .945, for the differences in shedding
from weeks 2 through 6, Fisher’s exact test).
Identification of nt 472 point mutations. After digestion at
the EcoRI site, the PCR product from the nonmutant construct
produced a visible 253-bp band and a 37-bp segment that was
run off during agarose gel electrophoresis. The PCR product from
the mutant construct was not digested by EcoRI and produced
a single 290-bp band (figure 3). The mixture of mutant and
nonmutant sequences replicated from stool samples often resulted in 2 distinct bands at 290- and 253-bp lengths (figure 3).
The MP among OPV3-positive stool samples. MAPREC
was used to calculate the MP for 37 of the 44 OPV3-positive
samples. The other 7 OPV3-positive samples were not used,
because they displayed unacceptably high background. The MP
of the samples obtained during the first 2 days after vaccination
was 10%–60%. The MP of the samples obtained on days 5–7
was 60%–100%. All the samples collected from day 14 to the
end of the sample-collection period had MPs close to 100%
(figure 4).
The 37 samples for which the MP could be estimated came
from 16 children (1–4 samples/child). Since measurements
from the same patient were not independent, simple trend
analysis was not valid. The mean MP in the first week after
administration of OPV (early MP) and the mean MP in weeks
2–6 (late MP) were calculated for each patient. The mean early
MP was 59.0% 8.8% (n p 11 children), whereas the mean
late MP was 98.9% 0.5% (n p 10 children). The 2 means
were significantly different (P p .001 , t test), and this difference
were lower (mean early MP, 0.590) and, thus, were subject to
a significant correction by calculating the AP. The mean MP
of the samples obtained during the first week after administration of OPV was 0.590, compared with the mean AP of
0.540 for the same time period. Therefore, when partial digestion is taken into account, the difference between the nt 472
MP at the first week after OPV administration and the MP at
later weeks becomes even more pronounced.
DISCUSSION
Figure 3. Eighteen-lane sample gel containing control samples and samples obtained from patients. Nonmutant virus subjected to EcoRI digestion
will produce a single 253-bp band. Mutant virus is resistant to EcoRI
digestion, and a single 290-bp band is shown. Mixtures of mutant and
nonmutant sequences will produce both 253- and 290-bp bands. Lane 1,
Nonmutant virus control construct, undigested; lane 2, nonmutant virus
control construct, digested; lane 3, mutant virus control construct, undigested; lane 4, mutant virus control construct, digested; lane 5, patient
sample 1, undigested; lane 6, patient sample 1, digested; lane 7, patient
sample 2, undigested; lane 8, patient sample 2, digested; lane 9, 100-bp
ladder; lane 10, 25-bp ladder; lane 11, patient sample 3, undigested; lane
12, patient sample 3, digested; lane 13, patient sample 4, undigested; lane
14, patient sample 4, digested; lane 15, patient sample 5, undigested; lane
16, patient sample 5, digested; lane 17, patient sample 6, undigested; and
lane 18, patient sample 6, digested.
was made more pronounced if only preparations with a high
LR were used (mean early MP, 50.0% 10.1%; mean late MP,
99.9% 0.1%; P ! .001).
Accounting for faulty undigested fragments. MPs were calculated assuming that PCR products not containing the point
mutation were completely digested by EcoRI. That was not always
the case. A close examination of digested nonmutant control
lanes occasionally revealed a low-intensity band in the 290-bp
region. The mean proportion of incompletely digested control
nonmutant constructs ranged from 10.2% 1.8%, for samples
with a high LR, to 12.6% 1.5%, for samples with a low LR.
This range was significantly different from 0, but not from 10%.
This finding is consistent with those of previous studies documenting incomplete digestion of DNA by PCR [35].
To adjust for incomplete digestion, which would falsely increase the observed MP, we developed the adjusted MP (AP).
The AP cannot be greater than the MP, and it equals the MP
only when both are exactly 1. Moreover, when the MP is close
to 1, the correction expressed in AP is negligible. The MPs for
weeks 2–6 were close to 1 (mean late MP, 0.989), and the AP
for the same time period was also very close to 1 (mean late
AP, 0.988). Thus, adjusting for incomplete digestion of PCR
products had minimal effect on the MPs for weeks 2–6. However, the MPs for the first week after administration of OPV
Polioviruses are among the best-studied human viruses, yet the
pathogenesis of polio vaccine–associated neurovirulence is not
well characterized. In the present study, we have found evidence
that OPV3 shedding patterns are strongly associated with the
development of a single point mutation related to OPV3 VAPP,
with statistically significant increases in shedding of VAPP-associated neurorevertant viruses after the first week of administration of OPV. The present study is the first to present such
data using stringent in vivo methods to isolate large populations
of poliovirus vaccine genomes. Direct extraction and RT-PCR
were used to detect a highly conserved OPV3 genome segment
from stool samples. MAPREC was then used to determine the
relative amount of viral sequences containing the OPV3-specific
UrC point mutation at nt 472 associated with neurovirulent
infection.
Although tissue culture is considered to be a standard
method of virus isolation and purification, it is becoming obsolete for the rapid detection of enteroviruses [36, 37]. The use
of tissue-cultivation techniques for the identification of viruses
requires a substantial quantity of live virus to produce a single
plaque on cell monolayers. For polioviruses in particular, as
many as 1000 live viruses are required for the creation of 1
plaque [38, 39]. Another consequence of tissue cultivation is
Figure 4. The UrC nt 472 mutant proportions of the oral poliovirus
vaccine (OPV) type 3–positive stool samples obtained from the 28 study
infants for 6 weeks after administration of OPV. Each dot represents 1
OPV3-positive stool sample.
Detection of Sabin Poliovirus Mutants by RNA Extraction and MAPREC • JID 2004:190 (15 July) • 413
the subsequent attenuation that occurs when viruses are subjected to passage through cell lines [20–23, 40]. This attenuation
may amplify or mask in vivo mutations, interfering with estimation of the proportion of mutants. To avoid these limitations, we modified a GuSCN extraction method for the specific detection of OPV3 [24, 29]. In a previous study of the
development of this technique, we found GuSCN direct extraction, in conjunction with RT-PCR, to be a reliable method
of isolating and amplifying a broad population of OPV3 viral
sequences in human stool samples [24]. It does not require
passage through live cells and therefore prevents the introduction of in vitro genetic modifications.
Previous investigations have studied the development of neurorevertant vaccine polioviruses in clinical samples [41–43]. In
2 of these studies, multiple clinical samples were available for
sequencing [42, 43]. Both studies used tissue culture and plaque
purification to analyze the development of OPV1, OPV2, and
OPV3 VAPP–associated point mutations. In the first study, a
single stool sample from each patient was obtained 1 month
after vaccination. Four OPV3 isolates were identified, and all
contained the UrC point mutation at nt 472. In the second
study, stool samples were obtained in 3 time periods after OPV
challenge: 1–10 days, 11–30 days, and 31–60 days. All 34 OPV3
isolates from the 3 collection periods expressed the neurorevertant UrC point mutation at nt 472. Together, these studies
emphasize the prevalence of OPV3-associated nt 472 neurorevertants in stool samples after administration of OPV.
In another study, a direct-extraction technique similar to
ours was compared to tissue-culture methods, to determine the
persistence of shedding of OPV1, OPV2, and OPV3 from stool
samples obtained from infants after the first OPV dose [44].
The investigators noted that direct extraction and RT-PCR recovered poliovirus at a higher rate than did tissue culture,
providing further support for direct-extraction techniques as
useful alternatives to tissue-cultivation methods. In addition,
that study confirmed the comparable sensitivity of extraction
methods in uniformly detecting the persistence of viral shedding for up to 8 weeks, compared with tissue culture. This is
similar to our previous results by use of tissue-cultivation methods to identify vaccine polioviruses, in which shedding of OPV3
in clinical samples was documented for up to 8 weeks after
administration of OPV [29].
In the present study, we have observed a clear, biphasic pattern of OPV3 shedding lasting throughout the 6-week study
period. On day 3, 35% of the infants were shedding OPV3.
Shedding decreased to 4% on day 6 and 6% on day 7. By the
beginning of the second week, 20% of the infants were again
shedding OPV3, and viral shedding remained at that level until
the end of the study. Using MAPREC to determine the relative
presence of mutant to nonmutant sequences in our clinical
samples, we were able to estimate the rate of development of
414 • JID 2004:190 (15 July) • Martinez et al.
the UrC point mutation at nt 472. During the first week after
vaccination, the MP increased from 0% to nearly 100%, and,
from day 14 until the end of the study period, the MP remained
close to 100%. The biphasic shedding pattern and the coinciding development of mutant sequences suggest that the replication dynamics of OPV3 occurring during the first week after
vaccination produced a strong selective pressure for development of the nt 472 mutant.
The UrC point mutation at nt 472 occurs in the 5 nontranslated region of the poliovirus genome. Although this is a
nonstructural coding region, it is an important locus, coding
for the internal ribosomal entry site (IRES) [45, 46]. A study
by Gromeier et al. showed that substitution of poliovirus IRES
by a rhinovirus IRES eliminates neurovirulence in the resulting
intergeneric poliovirus recombinants [45]. This result was further explored in a subsequent study, showing that 2 adjacent
stem-loop structures within the IRES seem to cooperatively
confer neuropathogenicity [46]. Given this evidence, it appears
that mutations occurring within the 5 noncoding region may
affect the replication of poliovirus in the gastrointestinal tract
and may play a role in modulating neuropathogenicity. The
dramatic increase in OPV3 shedding seen in conjunction with
the development of increasing levels of the nt 472 point mutation clearly indicate that selective pressures favor this mutation. It also appears that the attenuated strain is unstable in
vivo and that the nt 472 point mutation readily reverts to a
neuropathogenic sequence. However, the mechanism leading
to increased neurovirulence remains unclear.
The ability to detect distinct point mutations in vivo allows
us to define the dynamics of OPV replication after administration
of vaccine. Because of the concern that specific poliovirus vaccine
mutations are associated with VAPP, these results have direct
relevance to the pathogenesis of poliomyelitis in humans. However, there are limitations in the present study that must be
addressed. Future studies should expand the number of study
subjects to include children who have received only OPV in the
primary vaccination series and should include analysis of interactions among the 3 Sabin poliovirus vaccine serotypes and the
effect on VAPP-associated point mutations for all 3 serotypes.
Furthermore, studies using RNA extraction and MAPREC methods could provide information on the dynamics of household
and community transmission of OPV, with modifications to include assays for the detection of viable virus.
Several reports indicate that vaccine polioviruses continue to
circulate, even in well-vaccinated areas [47–49], illustrating a clear
link to vaccine viruses in the propagation and persistence of poliovirus in human communities. They also highlight the need for
continued surveillance of circulating polioviruses. Determining
the difference between the genomic modifications of OPV in the
human intestinal tract and in tissue culture is important in developing poliovirus eradication strategies. The ability to rapidly
determine poliovirus mutations for all serotypes is essential in
defining the circulation patterns of poliovirus. The present study
focused on OPV3 because, in the United States, it is the most
common cause of VAPP in immunologically healthy individuals
[15]. Future studies of OPV1 and OPV2 neurorevertant point
mutations in vivo will further our understanding of poliovirus
pathogenesis.
Acknowledgments
We thank Marvin Sommer and John Whitin for their technical assistance.
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