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Journal of Virology, September 2000, p. 8434-8443, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Natural Genetic Exchanges between Vaccine and Wild
Poliovirus Strains in Humans
Sophie
Guillot,1
Valérie
Caro,1
Nancy
Cuervo,1
Ekaterina
Korotkova,2
Mariana
Combiescu,3
Ana
Persu,3
Andrei
Aubert-Combiescu,3
Francis
Delpeyroux,1 and
Radu
Crainic1,*
Molecular Epidemiology of Enteroviruses,
Institut Pasteur, Paris, France,1
Belozerski Institute, Moscow State University, Moscow,
Russia,2 and Institut Cantacuzino,
Bucharest, Romania3
Received 20 March 2000/Accepted 23 June 2000
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ABSTRACT |
In a previous study of poliovirus vaccine-derived strains isolated
from patients with vaccine-associated paralytic poliomyelitis (VAPP)
(9, 11), we reported that a high proportion (over 50%) of
viruses had a recombinant genome. Most were intertypic
vaccine/vaccine recombinants. However, some had restriction fragment
length polymorphism (RFLP) profiles different from those of poliovirus
vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin.
To identify the parental wild donor of these sequences, we used RFLP
profiles and nucleotide sequencing to look for similarity in the 3D
polymerase-coding region of 61 wild, cocirculating poliovirus isolates
(43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other
four vaccine/wild recombinants, the wild parent could not be
identified. The possibility that the wild sequences were of a
non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an
isolated phenomenon. We also found wild polioviruses (2 of 15) carrying
vaccine-derived sequences in the 3' moiety of their genome. All these
results suggest that genetic exchanges with wild poliovirus and perhaps
with nonpoliovirus enteroviruses, are also a natural means of evolution
for poliovirus vaccine strains.
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INTRODUCTION |
Poliovirus (PV) is a
human enterovirus belonging to the Picornaviridae family.
The poliovirion is an icosahedral particle incorporating 60 copies each
of four virus-specified polypeptides: VP1, VP2, VP3, and VP4. This
protein coat encloses a single-stranded RNA genome of positive
polarity, 7,500 nucleotides in length. The single large open reading
frame is flanked by 5' and 3' extremities consisting of untranslated sequences.
PV, the causal agent of poliomyelitis, has three serotypes that do not
cross-react in neutralization tests with type-specific sera. If panels
of neutralizing monoclonal antibodies are used, antigenic differences
are observed between the strains of the three serotypes. However, these
differences are invariably confined to the limits of each serotype
(5, 17, 23). This has made possible the efficient
prophylaxis of poliomyelitis using two vaccines: the inactivated
poliovirus vaccine (IPV) and the oral poliovirus vaccine (OPV),
both of which contain all three PV serotypes.
The OPV consists of live attenuated Sabin strains obtained from wild
strains (29). The OPV strains actively multiply in the gut
of vaccinees, thereby eliciting a strong, long-lasting immune response
(30). The digestive immunity induced by OPV prevents or
limits reinfection in humans, thereby preventing natural PV
transmission (13). This property has made OPV the main tool for poliomyelitis eradication (39).
Sabin PV strains have a generally good safety record in vaccine use.
However, the selection of variants with increased neurovirulence constitutes a real problem with respect to vaccine safety. There is a
low but constant risk of vaccine-assisted paralytic poliomyelitis (VAPP) with the use of OPV. The overall incidence of VAPP in the United
States was found to be one case per 2.4 million doses distributed; for
immunocompetent children receiving their first dose of OPV, the
incidence was estimated at one case per 750,000 doses (28, 35). VAPP is most frequently associated with type 2 and 3 vaccine strains and rarely with type 1 vaccine strains (2, 24, 35). The risk of VAPP has increased by a factor of 10 to 14 in Romania over
the last three decades (6, 34). To reduce this risk and to
facilitate the transition to the exclusive use of IPV, a sequential
vaccination schedule in which IPV is followed by OPV was recently
introduced in the United States (1).
Sabin vaccine strains are subject to genetic variation during
their multiplication in humans. Indeed, following their
administration to humans, mutants bearing specific nucleotide
changes are rapidly isolated, probably due to selection pressure
exerted by factors such as temperature, target host cells, neutralizing
antibodies, and other unknown factors (reviewed in reference
23). Loss of the original attenuated phenotype of
OPV strains has been attributed to single- or multiple-nucleotide
substitutions at precise sites in the genome. Natural genetic
recombination in PV, another mechanism of variation in Sabin strains,
was demonstrated many years ago (14, 32, 33) (for reviews,
see references 4 and 19). Sabin
vaccine PV-derived strains with recombinant intertypic genomes have
been found to occur naturally (17, 22) and to be selected frequently in the gut of VAPP patients (9, 11, 21). In some
cases, vaccine/wild (V/W) PV recombinants have been found, in which
vaccine-specific segments of the Sabin virus genome have been replaced
by nonvaccine sequences derived from wild PVs or, perhaps, from
non-polio enteroviruses (NPEVs) (10, 19, 21, 30).
We previously used a combined analysis of two distant polymorphic
segments of the viral genome to analyze a large number of OPV-derived
strains isolated from VAPP cases in Romania. We detected a large number
of vaccine/vaccine (V/V) recombinant strains (9). Most were
easily recognized as intertypic V/V recombinants in restriction
fragment length polymorphism (RFLP) tests, but we also observed many
vaccine strains with genomic segments that gave atypical restriction
patterns. These segments were thought to be derived from modified Sabin
strains, wild PV, or from NPEVs. One of these recombinants was found to
be a double recombinant containing wild sequences throughout the genome
except in the capsid-encoding region (10). In this study, we
further analyzed the genetic characteristics of these modified
OPV-derived strains and assessed the occurrence of such strains in
cases of VAPP. Nonvaccine sequences were found to be present in 6% of
VAPP strains (V/W recombinants). We analyzed wild cocirculating PV
strains in the search for the nonvaccine parentals of the V/W
recombinants. We also detected OPV-derived sequences in the genomes of
these wild strains (9% of cases). These results suggest that genetic exchanges with wild PVs (and perhaps with NPEVs) are a common mechanism
of evolution for vaccine PV. We present here a detailed analysis of the
five V/W Romanian recombinants and a sixth strain isolated in Belarus.
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MATERIALS AND METHODS |
Cells and viruses.
HEp-2c and simian Vero cells were grown
in monolayers in Dulbecco modified Eagle medium supplemented with 5%
fetal calf serum. The PV vaccine viruses, Sabin 1, 2, and 3, were
obtained from the World Health Organization [Behringwerke (S0+1)]
"master seeds." The second passage at 34°C in HEp-2c cells of the
original seed was used to prepare viral stocks. The reference virulent
strains, PV1/Mahoney, PV2/MEF1, and PV3/Leon/37, were maintained in our laboratory and grown at 37°C in HEp-2c cells. The nomenclature used
for strains in this study includes the PV serotype (P1, P2, or P3),
origin (W, wild; V, vaccine-derived), laboratory index number (three
digits), and year of isolation (two digits). Most of the PV strains
studied here were isolated in Romania between 1980 and 1990.
Viruses were isolated from clinical specimens (generally stool
specimens, but also nasopharyngeal swabs, samples of cerebrospinal fluid, or central nervous system tissue obtained at autopsy) from patients with poliomyelitis by using primary cultures of monkey kidney
cells. The viruses were cultured further and titrated with HEp-2c
cells. Viral stocks were prepared by clarifying the supernatant of
infected cells by centrifugation for 20 min at 1,000 × g
and were stored at 
70°C. For neurovirulence tests, viruses were
concentrated by ultracentrifugation at 30,000 rpm in a Kontron 50.38 rotor for 2 h and resuspended in phosphate-buffered saline
supplemented with Ca++ and Mg++.
Virus identification.
Viruses were identified and typed by
using a standard neutralizing assay with type-specific neutralizing
antisera, as previously described (9, 11). Intratypic
differentiation between W and V strains was carried out with
strain-specific monoclonal antibodies (5). These antibodies
were also used to separate homotypic strains from samples containing
mixtures of V and W PVs. Virus origin (W or V) was systematically
confirmed by RFLP assay (see below) (3). All strains
analyzed in detail in this study were cloned by plaque purification.
Virus stocks were prepared by one or two passages in vitro after
cloning. If necessary, viruses were separated from mixtures by endpoint
dilution in the presence of heterotype-specific neutralizing antisera
and by plaque purification.
RFLP assay.
Recombinant genomes were detected with a double
RFLP assay that has been described elsewhere (9). Briefly,
two distant regions of the viral genome were subjected to PCR
amplification followed by restriction enzyme digestion. The first
region corresponded to nucleotides (nt) 2402 to 2881 (RFLP 1 assay) in
the VP1 capsid-encoding region, and the second corresponded to nt 6086 to 6376 in the 3D polymerase-coding region (RFLP 3D1 assay). The RFLP
profiles obtained with three different restriction enzymes from strains isolated in the field were compared with those of the original Sabin
strains. If they were found to differ, the RFLP profiles of the field
strains were compared with those of the wild strains tested.
We developed new RFLP assays to screen the entire genome for the
recombination junction in the recombinant strains. Restriction
enzymes
were selected to differentiate the three Sabin strains
over the entire
length of the genome. The pairs of primers and
the restriction enzymes
used in RFLP assays are listed in Table
1
(some of the primers presented have been used in previous studies
[
3,
9,
11,
27]).
Viral RNA was reverse transcribed, and the resulting cDNA was amplified
by PCR as previously described (
3) but with a few
modifications. Viral supernatant (1 µl), RNasin (20 U), and antisense
primer (10 pmol) were heated at 80°C for 5 min and annealed by
incubation at 42°C for 5 min. A mixture of 4 µl of transcriptase
buffer (5×), 1 µl of deoxynucleoside triphosphates (10 mM each),
1 U
of avian myeloblastosis virus reverse transcriptase, and distilled
water to make a final volume of 20 µl was added to the annealed
template-RNA solution, and transcription was allowed to occur
at 42°C
for 30 min. The reverse transcription (RT) products were
heated at
95°C for 5 min for denaturation and placed immediately
on ice. For
primers UC14, UC16, and UC10, the temperature for
annealing and RT was
increased to 50°C.
The PCR products were digested with restriction enzymes, and the
digestion products were resolved by electrophoresis as previously
described (
3,
9). The amplified fragment was named according
to the corresponding region of the PV
genome.
Nucleotide sequence analysis.
PCR products were sequenced
with the primers used for the RFLP tests. Before being sequenced, PCR
products were purified either directly after amplification on Qiaquick
spin columns (Qiagen) or by isolating bands from low-melting-point
agarose gels after electrophoresis by standard techniques
(31).
PCR products were sequenced by the dideoxynucleotide method with the
Sequenase kit, version 2.0 (U.S. Biochemicals) as previously
described
(
9). We also used the BigDye Terminator Cycle Sequencing
Ready Reaction kit, following the procedure recommended by Applied
Biosystems, Perkin-Elmer. Sequences were aligned with the CLUSTAL
W
program, version 1.6 (
37). The GenBank DNA sequence library
was screened for similar sequences with the FastA program, version
3.0 (
25). Dendrograms were constructed based on sequence
similarity,
using the programs included in the PHYLIP package, version
3.5
(
7). The distance matrix was calculated by the Kimura
two-parameter
method, using DNADIST. Three reconstruction was performed
with
the KITSCH program of the PHYLIP package. In the dendrogram, the
distance along the abscissa to the node connecting any two strains
is a
measure of the sequence divergence between those two strains.
The tree
was drawn using NJplot (
26).
Neurovirulence.
The neurovirulence of field isolates was
compared with that of reference Sabin strains and wild reference
viruses using mice transgenic for the human PV receptor gene (PVR-Tg
mice) (kindly provided by Akio Nomoto and T. Nomura). The
intraperitoneal inoculation-mean healthy time (IP-MHT) test was used as
previously described (11), except that each virus strain was
tested in 12 mice instead of 6. Briefly, each mouse was inoculated IP
with 108 PFU of virus strain in 500 µl of
phosphate-buffered saline. The MHT was calculated for each virus as the
mean number of days for which inoculated mice presented no clinical
neurological signs (paresis, paralysis or prominent walking
instability, or death) during the 14 days of observation. In most
cases, the disease index ratio (or percentage) of diseased mice versus
inoculated mice was also calculated.
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RESULTS |
Detection of recombinant strains.
We carried out an exhaustive
search for recombinant genomes in 88 vaccine-derived PV strains
isolated in Romania from patients with VAPP (9, 11). All
patients from whom OPV-derived strains were isolated presented a
poliomyelitis syndrome with persistent acute flaccid paralysis and were
classified as having VAPP (all had received vaccine or had been in
contact with vaccinees) according to World Health Organization case
classification criteria (38). For each strain, two distant
regions of the genome were studied by RT-PCR and comparison of
restriction digestion profiles were studied by RFLP: the RFLP 1 and
RFLP 3D1 assays were used to analyze the 5' third of the VP1-coding
region and the 5' part of the 3D polymerase-coding region, respectively
(11). For some strains, RFLP analysis was extended to two
other genomic regions: the RFLP P2 assay analyzes almost all of the
2C-coding region and the RFLP 3D-3' assay analyzes the 3' end of the
genome (see Materials and Methods). In this way, it was also possible
to detect a multiple recombinant. The results of the screening of 88 V
strains isolated from VAPP cases in Romania between 1980 and 1990, including those previously published (9-11), are presented
in Table 2. None of the serotype 1 strains was found to have a recombinant genome. The proportion of
recombinants was 81% for serotype 2 and 80% for serotype 3. Serotype
2 intertypic V/V recombinants showed preferential recombination with
Sabin 1-derived sequences in the 3D polymerase-coding region and 3'
untranslated region (3'UTR) extremity of the genome (64%), whereas
serotype 3 recombinants showed preferential recombination with Sabin 2 partners (68%).
Most recombinants were found to be intertypic V/V recombinants, but
five Sabin 2 strain genomes contained nonvaccine (W) sequences,
as
shown by sequencing (see details below) and were therefore
identified
as V/W recombinants. For the 88 strains isolated in
Romania, V/W
recombinants accounted for 13% of the type 2 recombinants
and 5.7% of
all vaccine-derived strains tested (Table
2). Four
of the five V/W
recombinants were isolated during a period in
which wild PV was
circulating in the region, but one (P2-V/057/87)
was isolated 5 years
(59 months) after the last recorded isolation
of wild PV in Romania. We
also analyzed a PV strain (P2-V/236/66)
isolated in January 1966 in
Moghilev, Belarus, from a 2-year-old
unvaccinated patient with
paralytic poliomyelitis. This Sabin
2 strain also had a nonvaccine
sequence in the 3' part of the
genome, as shown by sequencing. Clinical
and epidemiological data
for patients from whom V/W recombinant strains
and putative wild
donor strains were isolated are presented in Table
3.
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TABLE 3.
Clinical and epidemiological data on poliomyelitis cases
from which V/W recombinant and cocirculating wild strains
were isolated
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Genomic structure of V/W recombinants.
All six V/W
recombinants examined in this study were derived from Sabin 2 virus, as
shown by their reaction with Sabin 2-specific neutralizing monoclonal
antibodies (data not shown). In the RFLP 1 assay, Sabin 2-specific
restriction patterns were detected in the VP1-coding region.
To locate the recombination junction, we first scanned almost all of
the genome of these recombinant strains by a multiple
RFLP technique
(see Materials and Methods). Once the region containing
the junction
had been determined, it was sequenced to define the
recombination site
more precisely. The junction was located to
within a few nucleotides,
depending on the nucleotide differences
between the vaccine and the
wild strains. The recombination junctions
were found to be different
for each V/W strain (Fig.
1).
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FIG. 1.
Genomic structure and neurovirulence of the V/W
recombinant strains. (A) The genetic organization of the PV genome with
the structural capsid proteins (VP1 through VP4) and the nonstructural
proteins (2A through 3D) is shown. The size of the genome in kilobases
is also indicated. Genomic fragments, obtained by RT-PCR and analyzed
by RFLP, are indicated by boxes, and the name of each RFLP assay is
given. (B) Schematic diagrams of the genomes of the Sabin 2 and
recombinant V/W strains are shown. The structure of each genome was
deduced from the RFLP assays shown in panel A and from partial DNA
sequencing. Sequenced regions are indicated in boxes.
 , Sabin 2 sequence;  , nonvaccine sequence. Sequences at the
recombination junctions are given and compared with Sabin 2 sequences.
The results of neurovirulence tests are indicated: MHT and the numbers
and percentages of diseased transgenic mice after inoculation are given
for six of the seven strains shown in panel B. (C) Percent nucleotide
identity between the six V/W recombinants for three different regions
of the genome. The sequences of the genome compared are indicated.
VP1-2A, nt 3295 to 3444; 2C, nt 4546 to 4895; 3D1, nt 6172 to 6371 (nucleotide positions are numbered according to the Sabin 2 sequence).
Sequences were compared pairwise using the CLUSTAL W program, and the
percent nucleotide identity values for the pairs of strains are
shown.
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No recombination event was detected in the capsid-encoding region,
except in a small segment at the 3' end of the VP4-coding
region in
strain P2-V/598/80 (
10). The recombination site of
strain
P2-V/263/66 was located between nt 3393 and 3432, near
the junction
between the VP1- and 2A-coding regions (nt 3385).
This accounts for the
lower-percentage nucleotide identity in
the sequenced VP1-2A region
(positions 3295 to 3444) between P2-V/263/66
and the other strains,
including Sabin 2 (Fig.
1). In most cases,
sequence comparisons for the
2C and 3D nonstructural protein-coding
regions indicated that the W
sequences in the 3' moiety of the
V/W recombinants were different in
each strain (Fig.
1). This
suggested that the V/W recombinants resulted
from recombination
with different wild donors. However, the nucleotide
sequence of
strain P2-V/553/80 was found to be 92% identical to that
of strain
P2-V/146/81 in the 2C region and 94% identical to that of
strain
P2-V/057/87 in the 3D1 region. This suggests that these
sequences
may have a common ancestor. The different partners and/or
different
sites of recombination indicated that the various V/W strains
resulted from independent recombination
events.
We estimated the circulation of the recombinant strains by determining
the number of mutations accumulated in the Sabin 2-specific
region of
their genomes. A total of 550 to 1,045 nt were sequenced
for each of
the V/W strains, including the VP1-2A, the VP2, and,
in some cases, the
2B (strain P2-V/553/80) or 3A-C (strain P2-V/057/87)
regions (Table
4). Only 0 to 2 substitutions were found
in each
of the six V/W strains examined. This indicates that the
recombinant
viruses had circulated or multiplied for only short periods
of
time before isolation (see Discussion).
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TABLE 4.
Percent nucleotide identities between parental Sabin 2 PV
sequences and the vaccine-derived sequences present in
V/W recombinants
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Neurovirulence of V/W recombinants.
We assessed the
pathogenicity of the V/W recombinants isolated from VAPP patients by
comparing the neurovirulence of these strains with that of their Sabin
2 parent. We used mice transgenic for the human PV receptor gene, which
have been shown to be susceptible to all three PV serotypes following
parenteral inoculation. The IP-MHT assay, which involves daily
follow-up of the clinical signs appearing in mice inoculated IP with
virus, was used as described in Materials and Methods. The MHT recorded
for each strain shown in Fig. 1 indicates that the five V/W Romanian
recombinant strains tested had lost their attenuated phenotype. An MHT
of 7.0 to 8.0, with a disease index (percentage of diseased mice) of 50 to 67% corresponds to moderate neurovirulence. In this test, Sabin 2 had an MHT of 14 days (with a disease index of 0%). This is the minimum value for neurovirulence (maximum attenuation). The MHT and
disease indices (in parentheses) obtained in several experiments with
neurovirulent reference strains were 3.3 (95%) for PV1/Mahoney, 5.8 (80%) for PV2/MEF1, and 2.0 (100%) for PV3/Leon/37.
Wild recombination partner.
We tried to identify the donor
strains for sequences in the 3' moiety of V/W recombinant strains by
aligning the W sequences (2C and 3D1 segments) with homologous PV or
NPEV sequences from a nucleotide sequence database (GenBank). The
results of this search are presented in Table
5. In all cases, for the 2C-coding region, the highest percentage of nucleotide sequence identity was that
with PVs (79.4% to 84.0%). Coxsackieviruses A21 (CA21) and A24 (CA24)
appeared to be the most similar NPEVs (70.0 to 74.1% nucleotide
identity). Comparison of the nucleotide and amino acid sequences
corresponding to the 2C region suggested that the W sequences were
derived from wild PVs and not from NPEVs. For the 3D polymerase-coding
region, both nucleotide (80.0 to 87%) and amino acid (95.5 to 98.5%)
sequence identities were too similar for significant differences to be
detected between the W sequences and PV, CA21, and CA24 sequences.
However, no CA21 or CA24 strains were isolated in Romania between 1970 and 1995.
We investigated whether the wild sequences present in the V/W
recombinants were typical of the cocirculating wild strains
by
analyzing the wild PVs isolated in Romania during the period
in which
the V/W recombinants were
isolated.
Five of the V/W recombinant strains, all Sabin type 2-derived
polioviruses, were isolated in Romania in 1980, 1981, or 1987.
In 1980 to 1982, two overlapping poliomyelitis outbreaks occurred:
one with a
wild type 2 (P2-W) PV, which occurred from March to
April 1980 (15 virologically confirmed cases), and the other with
a wild type 1 (P1-W)
PV, which occurred from March 1980 to July
1982 (133 virologically
confirmed cases). Of a total of 148 cases,
52 occurred in 1980, 85 in
1981, and 11 in 1982. Two sporadic
cases were recorded in 1980 in which
wild type 3 poliovirus (P3-W)
was isolated. A total of about 650 PV
isolates were obtained from
epidemic cases and their contacts in 1980 to 1982 in Romania.
No wild PV was isolated in Romania during the
10-year period from
August 1982 to March 1991, despite a high standard
of virological
surveillance. During this period, one case of acute
flaccid paralysis
was reported per 100,000 children under the age
of 15 years; 997
children were examined, and the mean annual
isolation rate for
NPEV was 7.8%. Curiously, the V/W strain
P2-V/057/87 was isolated
in the middle of this 10-year
period.
We tried to find the W partners from the sequences of the V/W
recombinants. We first screened 61 strains randomly chosen from
the
wild cocirculating PVs for similarities in RFLP profile in
the 3D
polymerase-coding region (RFLP 3D1 assay). These strains,
comprising 43 type 1 (P1-W), 16 type 2 (P2-W), and 2 type 3 (P3-W)
strains, were
grouped on the basis of similarity of RFLP 3D1 pattern
(see Materials
and Methods). Thus, the wild type 1 PV strains
were classified
into four groups, and the wild type 2 PV strains
were assigned to a
single group. The restriction profiles of two
wild type 1 and two wild
type 3 strains were found to be similar
to those of the Sabin 1 and
Sabin 2 strains, respectively. The
RFLP 3D1 profiles of strains
P1-W/004/81 and P1-W/522/81 were
similar to that of the V/W
recombinant, P2-V/598/80. No similarity
in RFLP 3D1 profile was found
between any of the other four V/W
recombinants and the wild PVs
tested.
The 3D1- and VP1-2A-coding regions were sequenced for representative
members of each of the RFLP 3D1 groups. To increase the
probability of
finding the W parents of V/W recombinants, a careful
case-by-case study
was carried out. We also sequenced the 3D1
and VP1-2A regions of
strains known to have been present in the
same geographical area and at
the same time as the patients from
whom V/W recombinants were isolated.
Phylogenetic trees were constructed
from all these sequences, including
those of the five V/W recombinants
and the three OPV strains. The
dendrograms are shown in Fig.
2.
With the
exception of strain P2-V/598/80, no significant similarity
in the 3D1
genomic region was found between the V/W recombinants
and the wild
cocirculating PV strains tested. This suggests that
the wild donor of
four of the V/W recombinants may not be a wild
PV. However, we cannot
exclude the possibility that the wild partners
escaped at the first
step in the screening process, which involved
grouping strains
according to their RFLP 3D1 pattern.
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FIG. 2.
Dendrogram showing the relatedness of strains based on
the nucleotide sequences of the VP1-2A genomic region and those of the
3D polymerase-coding region. A schematic diagram of the PV genome (open
box) and the sequenced genomic regions (black boxes) is shown; VP1-2A
(positions 3295 to 3444) and 3D1 (positions 6172 to 6371) sequences
were used for this analysis. Sequencing data were analyzed using DNASIS
software, and the dendrogram was generated using the KITSCH program
from the PHYLIP package. The VP1-2A sequences are grouped according to
serotype: type 1,  ; type 2, ; type 3,  . The same
code is used for the 3D1 sequences. Vaccine, wild, and V/W recombinant
strains are shown in italics, boldface, and outlined capitals,
respectively. Percent nucleotide identity was estimated from the values
obtained for nucleotide identity (CLUSTAL W) and from the genetic
distances (KITCH) used for building the dendrogram.
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The dendrograms shown in Fig.
2 also confirm that two P1-W
strains (P1-W/639/80 and P1-W/640/80) contain Sabin 1-specific
sequences in the 3D1 segment of their genome (W/V recombinants).
The
recombination junction between wild and vaccine sequences
were
located for both strains around nt 5285. The vaccine-derived
nucleotide
sequences of these strains diverged by 1.0 and 2.0%
from those of the
original Sabin 1 strain, respectively, indicating
that either that
donor Sabin strain or the recombinant virus must
have been
circulating for several months before
isolation.
Wild PV partner of the V/W recombinant
P2-V/598/80.
Strain P2-V/598/80 has been shown to be a type
2 V/W recombinant in which the only conserved vaccine-derived sequence
is the capsid-coding region studied (10). Almost all of
the 5'UTR, the nonstructural protein-coding region, and the 3'UTR have
been replaced by wild sequences. In this study, we looked for the donor of the W nucleotide sequences in the 3D1 region and found a high degree
of sequence similarity to two wild PV strains, P1-W/004/81 and
P1-W/522/81, isolated at the beginning and end of 1981, respectively (Fig. 3). These two wild PV strains were
isolated from distant regions of Romania. This strongly suggests that
one of these P1-W epidemic strains or one of their ancestors was the
donor of the 3D1 sequence present in the P2-V/598/80 V/W recombinant
strain. With 97, 94, and 99% sequence identity in the 5'UTR, 2C, and
3D regions, respectively, the two P1-W strains appear to have closely related genomes. The low degree of nucleotide identity between the V/W
recombinant and the two P1-W strains in the 5'UTR (87 to 88%) and in
the 2C (77 to 78%) regions indicates that at least one other virus
donated these sequences. Strain P2-V/598/80 therefore probably has a
four-part recombinant genome resulting from genetic exchanges between
the Sabin 2 strain (capsid), at least one wild (PV or NPEV) partner
(5'UTR- and 2C-coding regions), and another wild PV (3Dpol
region).
View larger version (20K):
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|
FIG. 3.
Sequence similarities between the P2-V598/80 V/W
recombinant strain and two cocirculating wild PVs. A schematic diagram
of the genetic organization of the PV genome is shown. The 5'UTR and
3'UTR regions, the genomic regions encoding structural capsid proteins
(VP1 through VP4), and those encoding nonstructural proteins (2A
through 3D) are shown. The size of the genome in kilobases is also
indicated. A schematic diagram of the genome of P2-V/598/80, of the
cocirculating strains, and of P2-V/Sabin is given. Sequenced regions
are indicated: Sabin 2,  ; nonvaccine
sequence, . The sequenced parts of the genome compared
are indicated: 5' UTR, positions 220 to 468; VPI-2A, positions 3295 to
3444; 2C, positions 4546 to 4895; 3D1, positions 6172 to 6371. The
percent nucleotide identity between P2-V/598/80, the two epidemic wild
PVs, and the P2-V Sabin sequences was calculated after alignment with
CLUSTAL W.
|
|
The common ancestor of the closely related P1-W/004/81 and P1-W/522/81
or of another P1-W strain probably evolved by recombination.
Indeed,
the VP1-2A regions of P1-W/004/81 and P1-W/522/81 are
closely related
to all the other wild type 1 strains isolated
during the outbreak.
However, they fall into a different group
if the 3D
pol
region is considered (Fig.
2).
| |
DISCUSSION |
The analysis of PV strains isolated from vaccinees is an excellent
way of studying the evolution of enteroviruses in humans. Studies of
PVs isolated from patients with VAPP have demonstrated a high frequency
of genetic recombination between OPV strains (9-11, 17, 21,
22). A high frequency of intertypic vaccine-derived recombinants
was also found in this study by analyzing a total of 88 vaccine-derived
PV strains isolated from VAPP cases in Romania. Sequencing combined
with RFLP analysis for four different genomic fragments in the regions
encoding proteins VP1, 2C, and the 3D polymerase and for the 3'
extremity of the genome showed that 76% of the strains implicated in
these cases of VAPP (mostly types 2 and 3) had recombinant genomes.
Similar results have been obtained for OPV strains isolated from
healthy vaccinees (N. Cuervo et al., unpublished results).
Most of these recombinants were V/V recombinants, but five were shown
to contain nonvaccine genomic segments and were classified as V/W
recombinants. For one, the nonvaccine parent was identified among the
known cocirculating wild PVs. For the others, the wild parent has not
yet been identified and the possibility that the wild sequences
originate from an NPEV cannot be excluded. Wild PV or NEPV sequences
were detected in 6% of the VAPP strains studied. We analyzed wild
cocirculating PV strains in an attempt to identify the nonvaccine
parents of the V/W recombinants. We detected vaccine-derived sequences
in 2 of 15 wild strains. Our findings strongly suggest that there is
frequent interchange of genetic material between enteroviruses, whether
PVs or NPEVs. Vaccination with OPV creates ideal conditions for this
exchange by infecting the gut of the child with three different
enterovirus genotypes. In some cases there may already be infection, or
superinfection, with another enterovirus genotype.
Given the high frequency of recombinant genomes in the OPV strains
excreted by healthy vaccinees (22; N. Cuervo et al., unpublished results), their contacts in the community, and patients with VAPP (9, 11, 21; this study), it seems highly
likely that genetic recombination is involved in the natural evolution of Sabin strains. OPV strains are reputed to grow less efficiently in
the human gut than wild epidemic PVs. Due to the quasispecies properties of PV populations, a large number of variants are likely to
arise during the multiplication of vaccine strains in the human digestive tract. Two mechanisms may be responsible for this variation: mutations and recombination. Theoretically, recombination is a more
powerful mechanism of variation than mutation because it may transfer a
number of properties to the original virus in a single event. The
presence of variant populations of PV in the human gut provides a
wealth of material for the selection of variants that grow more efficiently.
We determined the neurovirulence of V/W recombinants in PVR-Tg mice and
found that none retained the attenuated phenotype of the original Sabin
2 PV. However, the degree of neurovirulence acquired was intermediate
between attenuation and the full neurovirulence of wild reference
strains. This suggests that recombination alone or in combination with
reverse mutations at the attenuating sites of Sabin 2 is not sufficient
to render the original vaccine virus highly neurovirulent. This
moderate increase in neurovirulence is, however, sufficient to allow
Sabin viruses to cause poliomyelitis in man, as demonstrated by the
identification of such strains as the etiological agent in certain VAPP
cases (12).
To estimate the length of time for which the V/W recombinant strains
had been circulating, we determined the number of mutations (as a
percentage of the total number of bases) accumulated in the
vaccine-derived segments. A large number of such mutations probably
indicates the circulation of vaccine viruses before and/or after the
recombination event. Very small numbers of mutations were found to have
accumulated in the Sabin 2-specific regions of the genomes of the six
V/W recombinants (from 0 to 0.2% mutated nucleotides). This indicates
that recombination probably occurred in the gut of the vaccine
recipient or in that of a contact before the disease. The number of
mutations accumulated in the Sabin 1 genome during multiplication in
humans, with or without disease transmission, has been estimated to be
close to 1% per year for the VP1-coding region (16, 18).
However, the frequency of variation may depend on genomic segment and
strain. Our results for the Sabin sequences suggest that the W/V
recombinants circulated or multiplied for several months before or
after recombination with wild PVs.
We cannot determine whether the W sequences in the 3D1 region
originated from wild PVs or from NPEVs, directly or via a wild PV,
until the donor virus has been positively identified. Four of the five
Romanian V/W recombinants were isolated during a period (1980 to 1982)
of active circulation of wild PV in Romania. In an attempt to determine
the origin of the wild sequences in V/W recombinants, we compared them
with homologous sequences from a nucleotide sequence database
(GenBank). PV rather than NPEV sequences were selected as the closest
match, favoring the hypothesis that silent circulating wild PVs rather
than NPEVs were the donors of the wild sequences of the V/W
recombinants. This led us to try to identify the wild parent among the
cocirculating epidemic PV strains. We looked for similarities in the
sequence of the 3D1 region between the recombinants and 61 of 148 wild
type 1, 2, and 3 poliovirus isolates. The W sequence in the 3D1 region of strain P2-V/598/80 appeared to be very similar to those of two type
1 wild PVs, isolated several months later in Romania (Fig. 3).
Phylogenetic analysis of the sequences suggested that the two wild PVs
and the donor of the sequence present in the V/W recombinant probably
have a common ancestor. A similar situation was found in China
(20), where two P1-V/W recombinants were found in which the
W donor was identified as a wild PV circulating in a geographically
narrow region. The demonstration that, during their natural
multiplication, the PV strains could "trap" sequences from
cocirculating wild PV may be used for an active detection of silent
wild PV transmission by screening for the V/W recombinants.
However, the donors of the other V/W recombinants described here have
not yet been identified among the 61 cocirculating wild PVs tested.
Surprisingly, one of the V/W recombinant strains (P2-V/057/87) was
isolated in the middle of a 9-year period (1983 to 1991) during which
no wild PV was isolated in the surrounding region, under conditions of
high-quality virological surveillance. Similarly, in Brazil, a V/W
recombinant was isolated from a VAPP case 2 years after the last
isolation of a wild PV on the American continent (8). This
suggests that a NPEV rather than a wild PV may have been the donor of
the W sequence of the recombinant. Although searches of GenBank mostly
showed the most similar sequence to be that of a wild PV, this may be
due to the small number of sequences from enteroviruses in the database
corresponding to the genomic regions studied here. In the
case of the W segments of the V/W recombinants, only the positive
identification of an NPEV donor of these sequences could completely
exonerate wild PV from being the recombination partner. There is no
experimental evidence that recombinants between PV and NPEV are viable.
However, there are findings suggesting that such recombinants circulate
in nature, as the remarkable similarity to PV of the 3' end of the CA21
or the CA24 reference strains shows (15, 36).
Thus, it is clear that OPV-derived PVs can acquire highly modified
genomes not only by mutation but also by genetic exchanges with vaccine
or wild PV, or even with NPEV. These PV vaccine-derived strains can
survive for long periods of time by prolonged excretion and/or by
natural transmission and may cause poliomyelitis in man. If PV
eradication is to be successful after OPV is discontinued, we must hope
that PV vaccine-derived strains will disappear before they can spread
in the growing nonimmune population. This should be carefully studied
before a general policy of discontinuing OPV immunization is implemented.
| |
ACKNOWLEDGMENTS |
This work was partly supported by grants to R.C. from the World
Health Organization (V26/181/107) and from the European Commission (Copernicus-CIPA CT94-0123 and Inco-Copernicus ERBIC 15 CT96-0912).
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Epidémiologie Moléculaire des Entérovirus, Institut
Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 1 4568 8763. Fax: 33 1 4568 8780. E-mail: craira{at}pasteur.fr.
| |
REFERENCES |
| 1.
|
Anonymous.
1997.
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).
Morb. Mortal. Wkly. Rep.
46:1-25[Medline].
|
| 2.
|
Assaad, F., and W. C. Cockburn.
1982.
The relation between acute persisting paralysis and poliomyelitis vaccine results of a ten-year inquiry.
Bull W. H. O.
60:231-238[Medline].
|
| 3.
|
Balanant, J.,
S. Guillot,
A. Candrea,
F. Delpeyroux, and R. Crainic.
1991.
The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.
Virology
184:645-654[CrossRef][Medline].
|
| 4.
|
Cooper, P. D.
1977.
Genetics of picornaviruses.
Comp. Virol.
9:133-207.
|
| 5.
|
Crainic, R.,
P. Couillin,
B. Blondel,
N. Cabau,
A. Boue, and F. Horodniceanu.
1983.
Natural variation of poliovirus neutralization epitopes.
Infect. Immun.
41:1217-1225[Abstract/Free Full Text].
|
| 6.
|
Crainic, R.,
M. Furione,
D. Otelea,
S. Guillot,
J. Balanant,
A. Aubert-Combiescu,
M. Combiescu, and A. Candrea.
1993.
Natural evolution of oral vaccine poliovirus strains, p. 371-390.
In
E. Kurstak (ed.), Measles and poliomyelitis. Springer-Verlag, Vienna, Austria.
|
| 7.
|
Felsenstein, J.
1993.
PHYLIP (Phylogeny Inference Package), version 3.5c.
Department of Genetics, University of Washington, Seattle, Washington.
|
| 8.
|
Friedrich, F.,
E. F. daSilva, and H. G. Schatzmayr.
1996.
Type 2 poliovirus recombinants isolated from vaccine-associated cases and from healthy contacts in Brazil.
Acta Virol.
40:27-33[Medline].
|
| 9.
|
Furione, M.,
S. Guillot,
D. Otelea,
J. Balanant,
A. Candrea, and R. Crainic.
1993.
Polioviruses with natural recombinant genomes isolated from vaccine-associated paralytic poliomyelitis.
Virology
196:199-208[CrossRef][Medline].
|
| 10.
|
Georgescu, M. M.,
F. Delpeyroux, and R. Crainic.
1995.
Tripartite genome organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus.
J. Gen. Virol.
76:2343-2348[Abstract/Free Full Text].
|
| 11.
|
Georgescu, M.-M.,
F. Delpeyroux,
M. Tardy-Panit,
J. Balanant,
M. Combiescu,
A. A. Combiescu,
S. Guillot, and R. Crainic.
1994.
High diversity of poliovirus strains isolated from the central nervous system from patients with vaccine-associated paralytic poliomyelitis.
J. Virol.
68:8089-8101[Abstract/Free Full Text].
|
| 12.
|
Georgescu, M. M.,
J. Balanant,
A. Macadam,
D. Otelea,
M. Combiescu,
A. A. Combiescu,
R. Crainic, and F. Delpeyroux.
1997.
Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.
J. Virol.
71:7758-7768[Abstract].
|
| 13.
|
Ghendon, Y., and S. E. Robertson.
1994.
Interrupting the transmission of wild polioviruses with vaccines: immunological considerations.
Bull. W. H. O.
72:973-983[Medline].
|
| 14.
|
Hirst, G. K.
1962.
Genetic recombination with Newcastle virus, polioviruses and influenza.
Cold Spring Harbor Symp. Quant. Biol.
27:303-308[Abstract/Free Full Text].
|
| 15.
|
Hughes, P. J.,
C. North,
P. D. Minor, and G. Stanway.
1989.
The complete nucleotide sequence of coxsackievirus A21.
J. Gen. Virol.
70:2943-2952[Abstract/Free Full Text].
|
| 16.
|
Kew, O. M.,
M. N. Mulders,
G. Y. Lipskaya,
E. E. daSylva, and M. A. Pallansch.
1995.
Molecular epidemiology of polioviruses.
Semin. Virol.
6:401-414[CrossRef].
|
| 17.
|
Kew, O. M., and B. K. Nottay.
1984.
Evolution of the oral poliovaccine strains in humans occurs by both mutation and intramolecular recombination, p. 357-367.
In
R. Chanock, and R. Lerner (ed.), Modern approaches to vaccines. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
|
| 18.
|
Kew, O. M.,
R. W. Sutter,
B. K. Nottay,
M. J. McDonough,
D. R. Prevots,
L. Quick, and M. A. Pallansch.
1998.
Prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient.
J. Clin. Microbiol.
36:2893-2899[Abstract/Free Full Text].
|
| 19.
|
Lai, M. M. C.
1992.
RNA recombination in animal and plant viruses.
Microbiol. Rev.
56:61-79[Abstract/Free Full Text].
|
| 20.
|
Li, J.,
L. B. Zhang,
T. Yoneyama,
H. Yoshida,
H. Shimizu,
K. Yoshii,
M. Hara,
T. Nomura,
H. Yoshikura,
T. Miyamura, and A. Hagiwara.
1996.
Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients.
Arch. Virol.
141:1047-1054[CrossRef][Medline].
|
| 21.
|
Lipskaya, G. Y.,
A. R. Muzychenko,
O. K. Kutitova,
S. V. Maslova,
M. Equestre,
S. G. Drozdov,
R. P. Bercoff, and V. I. Agol.
1991.
Frequent isolation of intertypic poliovirus recombinants with serotype 2 specificity from vaccine-associated polio cases.
J. Med. Virol.
35:290-296[Medline].
|
| 22.
|
Macadam, A. J.,
C. Arnold,
J. Howlett,
A. John,
S. Marsden,
F. Taffs,
P. Reeve,
N. Hamada,
K. Wareham,
J. Almond,
N. Cammack, and P. D. Minor.
1989.
Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees.
Virology
172:408-414[CrossRef][Medline].
|
| 23.
|
Minor, P. D.
1992.
The molecular biology of poliovaccines.
J. Gen. Virol.
73:3065-3077[Abstract/Free Full Text].
|
| 24.
|
Otelea, D.,
S. Guillot,
M. Furione,
A. A. Combiescu,
J. Balanant,
A. Candrea, and R. Crainic.
1993.
Genomic modifications in naturally occurring neurovirulent revertants of Sabin 1 polioviruses.
Dev. Biol. Stand.
78:33-38[Medline].
|
| 25.
|
Pearson, W. R., and D. J. Lipman.
1988.
Improved tools for biological sequence comparison.
Proc. Natl. Acad. Sci. USA
85:2444-2448[Abstract/Free Full Text].
|
| 26.
|
Perrière, G., and M. Gouy.
1996.
WWW-query: an on-line retrieval system for biological sequence banks.
Biochimie
78:364-369[Medline].
|
| 27.
|
Petitjean, J.,
M. Quibriac,
F. Freymuth,
F. Fuchs,
N. Laconche,
M. Aymard, and H. Kopecka.
1990.
Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic riboprobes.
J. Clin. Microbiol.
28:307-311[Abstract/Free Full Text].
|
| 28.
|
Prevots, D. R.,
R. W. Sutter,
P. M. Strebel,
R. E. Weibel, and S. L. Cochi.
1994.
Completeness of reporting for paralytic poliomyelitis, United States, 1980 through 1991. Implications for estimating the risk of vaccine-associated disease.
Arch. Pediatr. Adolesc. Med.
148:479-485[Abstract/Free Full Text].
|
| 29.
|
Sabin, A. B., and L. R. Boulger.
1973.
History of Sabin attenuated poliovirus oral live vaccine strains.
J. Biol. Stand.
1:115-118.
|
| 30.
|
Sabin, A. B.
1985.
Oral poliovirus vaccine: history of its development and use and current challenge to eliminate poliomyelitis from the world.
J. Infect. Dis.
151:420-436[Medline].
|
| 31.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed., vol. 1.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 32.
|
Sergiescu, D.,
C. A. Aubert, and R. Crainic.
1969.
Recombination between guanidine-resistant and dextran sulfate-resistant mutants of type 1 poliovirus.
J. Virol.
3:326-330[Abstract/Free Full Text].
|
| 33.
|
Sergiescu, D.,
F. Horodniceanu,
R. Klein, and R. Crainic.
1966.
Genetic transfer of guanidine resistance from type 2 to type 1 poliovirus.
Arch. Gesamte Virusforsch.
18:231-243[CrossRef][Medline].
|
| 34.
|
Strebel, P. M.,
C. A. Aubert,
N. N. Ion,
M. S. Biberi,
M. Combiescu,
R. W. Sutter,
O. M. Kew,
M. A. Pallansch,
P. A. Patriarca, and S. L. Cochi.
1994.
Paralytic poliomyelitis in Romania, 1984-1992. Evidence for a high risk of vaccine-associated disease and reintroduction of wild-virus infection.
Am. J. Epidemiol.
140:1111-1124[Abstract/Free Full Text].
|
| 35.
|
Strebel, P. M.,
R. W. Sutter,
S. L. Cochi,
R. J. Biellik,
E. W. Brink,
O. M. Kew,
M. A. Pallansch,
W. A. Orenstein, and A. R. Hinman.
1992.
Epidemiology of poliomyelitis in the United States one decade after the last reported case of indigenous wild virus-associated disease.
Clin. Infect. Dis.
14:568-579[Medline].
|
| 36.
|
Supanaranond, K.,
N. Takeda, and S. Yamazaki.
1992.
The complete nucleotide sequence of a variant of Coxsackievirus A24, an agent causing acute hemorraghic cunjunctivitis.
Virus Genes
6:149-158[CrossRef][Medline].
|
| 37.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
| 38.
|
World Health Organization.
1997.
Manual for poliovirus surveillance. WHO/EPI/GEN/97.01.
World Health Organization, Geneva, Switzerland.
|
| 39.
|
Wright, P. F.,
R. J. Kim-Farley,
C. A. de Quadros,
S. E. Robertson,
R. M. Scott,
N. A. Ward, and R. H. Henderson.
1991.
Strategies for the global eradication of poliomyelitis by the year 2000.
N. Engl. J. Med.
325:1774-1779[Medline].
|
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-
Gao, F., Nainan, O. V., Khudyakov, Y., Li, J., Hong, Y., Gonzales, A. C., Spelbring, J., Margolis, H. S.
(2007). Recombinant hepatitis C virus in experimentally infected chimpanzees. J. Gen. Virol.
88: 143-147
[Abstract]
[Full Text]
-
Mirand, A., Henquell, C., Archimbaud, C., Peigue-Lafeuille, H., Bailly, J.-L.
(2007). Emergence of recent echovirus 30 lineages is marked by serial genetic recombination events. J. Gen. Virol.
88: 166-176
[Abstract]
[Full Text]
-
Simmonds, P.
(2006). Recombination and Selection in the Evolution of Picornaviruses and Other Mammalian Positive-Stranded RNA Viruses. J. Virol.
80: 11124-11140
[Abstract]
[Full Text]
-
Romanenkova, N. I., Guillot, S., Rozaeva, N. R., Crainic, R., Bichurina, M. A., Delpeyroux, F.
(2006). Use of a Multiple Restriction Fragment Length Polymorphism Method for Detecting Vaccine-Derived Polioviruses in Clinical Samples. J. Clin. Microbiol.
44: 4077-4084
[Abstract]
[Full Text]
-
Gay, R. T., Belisle, S., Beck, M. A., Meydani, S. N.
(2006). An aged host promotes the evolution of avirulent coxsackievirus into a virulent strain. Proc. Natl. Acad. Sci. USA
103: 13825-13830
[Abstract]
[Full Text]
-
Noppornpanth, S., Lien, T. X., Poovorawan, Y., Smits, S. L., Osterhaus, A. D. M. E., Haagmans, B. L.
(2006). Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus.. J. Virol.
80: 7569-7577
[Abstract]
[Full Text]
-
Wierzchoslawski, R., Bujarski, J. J.
(2006). Efficient In Vitro System of Homologous Recombination in Brome Mosaic Bromovirus.. J. Virol.
80: 6182-6187
[Abstract]
[Full Text]
-
Simmonds, P., Welch, J.
(2006). Frequency and Dynamics of Recombination within Different Species of Human Enteroviruses. J. Virol.
80: 483-493
[Abstract]
[Full Text]
-
Lukashev, A. N., Lashkevich, V. A., Ivanova, O. E., Koroleva, G. A., Hinkkanen, A. E., Ilonen, J.
(2005). Recombination in circulating Human enterovirus B: independent evolution of structural and non-structural genome regions. J. Gen. Virol.
86: 3281-3290
[Abstract]
[Full Text]
-
Karakasiliotis, I., Paximadi, E., Markoulatos, P.
(2005). Evolution of a rare vaccine-derived multirecombinant poliovirus. J. Gen. Virol.
86: 3137-3142
[Abstract]
[Full Text]
-
Bukreyev, A., Huang, Z., Yang, L., Elankumaran, S., St. Claire, M., Murphy, B. R., Samal, S. K., Collins, P. L.
(2005). Recombinant Newcastle Disease Virus Expressing a Foreign Viral Antigen Is Attenuated and Highly Immunogenic in Primates. J. Virol.
79: 13275-13284
[Abstract]
[Full Text]
-
Arita, M., Zhu, S.-L., Yoshida, H., Yoneyama, T., Miyamura, T., Shimizu, H.
(2005). A Sabin 3-Derived Poliovirus Recombinant Contained a Sequence Homologous with Indigenous Human Enterovirus Species C in the Viral Polymerase Coding Region. J. Virol.
79: 12650-12657
[Abstract]
[Full Text]
-
Papaventsis, D., Siafakas, N., Markoulatos, P., Papageorgiou, G. T., Kourtis, C., Chatzichristou, E., Economou, C., Levidiotou, S.
(2005). Membrane Adsorption with Direct Cell Culture Combined with Reverse Transcription-PCR as a Fast Method for Identifying Enteroviruses from Sewage. Appl. Environ. Microbiol.
71: 72-79
[Abstract]
[Full Text]
-
Rakoto-Andrianarivelo, M., Rousset, D., Razafindratsimandresy, R., Chevaliez, S., Guillot, S., Balanant, J., Delpeyroux, F.
(2005). High Frequency of Human Enterovirus Species C Circulation in Madagascar. J. Clin. Microbiol.
43: 242-249
[Abstract]
[Full Text]
-
Suspene, R., Henry, M., Guillot, S., Wain-Hobson, S., Vartanian, J.-P.
(2005). Recovery of APOBEC3-edited human immunodeficiency virus G->A hypermutants by differential DNA denaturation PCR. J. Gen. Virol.
86: 125-129
[Abstract]
[Full Text]
-
Shimizu, H., Thorley, B., Paladin, F. J., Brussen, K. A., Stambos, V., Yuen, L., Utama, A., Tano, Y., Arita, M., Yoshida, H., Yoneyama, T., Benegas, A., Roesel, S., Pallansch, M., Kew, O., Miyamura, T.
(2004). Circulation of Type 1 Vaccine-Derived Poliovirus in the Philippines in 2001. J. Virol.
78: 13512-13521
[Abstract]
[Full Text]
-
Kalinina, O., Norder, H., Magnius, L. O.
(2004). Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation. J. Gen. Virol.
85: 1853-1857
[Abstract]
[Full Text]
-
Oberste, M. S., Maher, K., Pallansch, M. A.
(2004). Evidence for Frequent Recombination within Species Human Enterovirus B Based on Complete Genomic Sequences of All Thirty-Seven Serotypes. J. Virol.
78: 855-867
[Abstract]
[Full Text]
-
Colina, R., Casane, D., Vasquez, S., Garcia-Aguirre, L., Chunga, A., Romero, H., Khan, B., Cristina, J.
(2004). Evidence of intratypic recombination in natural populations of hepatitis C virus. J. Gen. Virol.
85: 31-37
[Abstract]
[Full Text]
-
Costa-Mattioli, M., Napoli, A. D., Ferre, V., Billaudel, S., Perez-Bercoff, R., Cristina, J.
(2003). Genetic variability of hepatitis A virus. J. Gen. Virol.
84: 3191-3201
[Abstract]
[Full Text]
-
Korotkova, E. A., Park, R., Cherkasova, E. A., Lipskaya, G. Y., Chumakov, K. M., Feldman, E. V., Kew, O. M., Agol, V. I.
(2003). Retrospective Analysis of a Local Cessation of Vaccination against Poliomyelitis: a Possible Scenario for the Future. J. Virol.
77: 12460-12465
[Abstract]
[Full Text]
-
Liu, H.-M., Zheng, D.-P., Zhang, L.-B., Oberste, M. S., Kew, O. M., Pallansch, M. A.
(2003). Serial Recombination during Circulation of Type 1 Wild-Vaccine Recombinant Polioviruses in China. J. Virol.
77: 10994-11005
[Abstract]
[Full Text]
-
Lukashev, A. N., Lashkevich, V. A., Ivanova, O. E., Koroleva, G. A., Hinkkanen, A. E., Ilonen, J.
(2003). Recombination in Circulating Enteroviruses. J. Virol.
77: 10423-10431
[Abstract]
[Full Text]
-
Brown, B., Oberste, M. S., Maher, K., Pallansch, M. A.
(2003). Complete Genomic Sequencing Shows that Polioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region. J. Virol.
77: 8973-8984
[Abstract]
[Full Text]
-
Boot, H. J., Kasteel, D. T. J., Buisman, A.-M., Kimman, T. G.
(2003). Excretion of Wild-Type and Vaccine-Derived Poliovirus in the Feces of Poliovirus Receptor-Transgenic Mice. J. Virol.
77: 6541-6545
[Abstract]
[Full Text]
-
Lindberg, A. M., Andersson, P., Savolainen, C., Mulders, M. N., Hovi, T.
(2003). Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species. J. Gen. Virol.
84: 1223-1235
[Abstract]
[Full Text]
-
Blomqvist, S., Bruu, A.-L., Stenvik, M., Hovi, T.
(2003). Characterization of a recombinant type 3/type 2 poliovirus isolated from a healthy vaccinee and containing a chimeric capsid protein VP1. J. Gen. Virol.
84: 573-580
[Abstract]
[Full Text]
-
Whitehead, S. S., Falgout, B., Hanley, K. A., Blaney, J. E. Jr., Markoff, L., Murphy, B. R.
(2002). A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3' Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys. J. Virol.
77: 1653-1657
[Abstract]
[Full Text]
-
Dahourou, G., Guillot, S., Le Gall, O., Crainic, R.
(2002). Genetic recombination in wild-type poliovirus. J. Gen. Virol.
83: 3103-3110
[Abstract]
[Full Text]
-
Martin, J., Samoilovich, E., Dunn, G., Lackenby, A., Feldman, E., Heath, A., Svirchevskaya, E., Cooper, G., Yermalovich, M., Minor, P. D.
(2002). Isolation of an Intertypic Poliovirus Capsid Recombinant from a Child with Vaccine-Associated Paralytic Poliomyelitis. J. Virol.
76: 10921-10928
[Abstract]
[Full Text]
-
Oprisan, G., Combiescu, M., Guillot, S., Caro, V., Combiescu, A., Delpeyroux, F., Crainic, R.
(2002). Natural genetic recombination between co-circulating heterotypic enteroviruses. J. Gen. Virol.
83: 2193-2200
[Abstract]
[Full Text]
-
Lawrence, J. G., Hatfull, G. F., Hendrix, R. W.
(2002). Imbroglios of Viral Taxonomy: Genetic Exchange and Failings of Phenetic Approaches. J. Bacteriol.
184: 4891-4905
[Abstract]
[Full Text]
-
Norder, H., Bjerregaard, L., Magnius, L. O.
(2002). Open reading frame sequence of an Asian enterovirus 73 strain reveals that the prototype from California is recombinant. J. Gen. Virol.
83: 1721-1728
[Abstract]
[Full Text]
-
Martin, J., Minor, P. D.
(2002). Characterization of CHAT and Cox Type 1 Live-Attenuated Poliovirus Vaccine Strains. J. Virol.
76: 5339-5349
[Abstract]
[Full Text]
-
Kalinina, O., Norder, H., Mukomolov, S., Magnius, L. O.
(2002). A Natural Intergenotypic Recombinant of Hepatitis C Virus Identified in St. Petersburg. J. Virol.
76: 4034-4043
[Abstract]
[Full Text]
-
Marturano, J., Fiore, L.
(2002). Investigation of the Presence of Recombinant Polioviruses in the Hit Population in Albania during the 1996 Outbreak. J. Clin. Microbiol.
40: 316-317
[Full Text]
-
Skiadopoulos, M. H., Surman, S. R., Riggs, J. M., Collins, P. L., Murphy, B. R.
(2001). A Chimeric Human-Bovine Parainfluenza Virus Type 3 Expressing Measles Virus Hemagglutinin Is Attenuated for Replication but Is Still Immunogenic in Rhesus Monkeys. J. Virol.
75: 10498-10504
[Abstract]
[Full Text]
-
Cuervo, N. S., Guillot, S., Romanenkova, N., Combiescu, M., Aubert-Combiescu, A., Seghier, M., Caro, V., Crainic, R., Delpeyroux, F.
(2001). Genomic Features of Intertypic Recombinant Sabin Poliovirus Strains Excreted by Primary Vaccinees. J. Virol.
75: 5740-5751
[Abstract]
[Full Text]
-
Liu, H.-M., Zheng, D.-P., Zhang, L.-B., Oberste, M. S., Pallansch, M. A., Kew, O. M.
(2000). Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China. J. Virol.
74: 11153-11161
[Abstract]
[Full Text]
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Abstract
Introduction
