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Applied and Environmental Microbiology, November 2000, p. 5087-5091, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Assessment of Poliovirus Eradication in Japan: Genomic Analysis
of Polioviruses Isolated from River Water and Sewage in
Toyama Prefecture
Kumiko
Matsuura,1,*
Mitsuhiro
Ishikura,1
Hiromu
Yoshida,2
Takashi
Nakayama,1
Sumiyo
Hasegawa,1
Shuji
Ando,1
Hitoshi
Horie,3
Tatsuo
Miyamura,2 and
Takashi
Kitamura1
Department of Virology, Toyama Institute of
Health, Toyama 939-0363,1 and Laboratory
of Enteroviruses, Department of Virology II, National Institute of
Infectious Diseases,2 and Japan
Poliomyelitis Research Institute,3 Tokyo, Japan
Received 17 May 2000/Accepted 29 August 2000
 |
ABSTRACT |
Seventy-eight poliovirus strains isolated from river water and
sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of
the viral genome. Of these isolates, 36 were identified as Sabin
vaccine strains, and 42 were identified as vaccine variant strains that
had less than 1.4% nucleotide divergence from the Sabin strains,
including 7 isolates with patterns different from those of Sabin
strains as determined by PCR-RFLP analysis. These findings suggest
that wild-type poliovirus was not circulating in Toyama Prefecture.
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TEXT |
The global polio eradication program
has progressed by the initiates of the World Health Organization
(4, 5). In Japan, the last outbreak of acute poliomyelitis
occurred in 1960, followed by a decline in outbreaks as the result of
the introduction of a live oral polio vaccine (OPV) for children in
1961. Since 1963, children have been protected by routine polio
vaccination by OPV, and the incidence of clinical polio infection has
been drastically reduced. The wild-type strains were detected in one
patient with poliomyelitis in 1980 and in two patients with nonacute
flaccid paralysis in 1984 and 1993, but since then no wild-type
polioviruses have been isolated (11). On such a background,
in order to verify polio eradication in Japan it is critical to confirm
that the wild-type strains have not been circulating in Japan by
characterizing the poliovirus strains excreted by inhabitants to
differentiate the possible introduction of wild virus from the agent of
vaccine-associated paralytic poliomyelitis. We predicted that the
genetic analysis of poliovirus isolated from environmental waters might
be a useful way of elucidating this. The viruses isolated from the
environmental waters, namely, river water and sewage, correlated well
with those excreted by inhabitants (13, 14, 15). Tambini et
al. have reported that it is important to study the ecology of
polioviruses excreted from human intestines into the environment at the
final stage of the polio eradication program (21). We have
examined the viruses in river water and sewage to monitor the viral
pollution in the environmental waters in Toyama Prefecture from October 1993 to September 1995. In the present study, the genetic
characteristics of the poliovirus strains isolated during this
examination were compared by sequence analysis of the VP3 and VP1
regions with the wild and Sabin vaccine strains of polioviruses.
Poliovirus isolates from river water and sewage.
Four sampling
stations (I, S, O, and G), as shown in Fig.
1, were chosen along the Itachi, Sembo,
and Oyabe Rivers and at a sewage disposal plant located alongside the
Oyabe River. The river water samples (approximately 700 to 800 ml),
squeezed from two cotton pads (50 g each) that were immersed in the
stream for 2 days at stations I, S, and O before sampling, were
collected twice a month from October 1993 to September 1995. The
sewage sample (1 liter) was collected from a sewage settling tank in station G at the same time as the river water samplings. These samples
were concentrated by using the filter adsorption and elution method
(13). A cellulose nitrate membrane filter (0.45- µm pore size, type TM2; Toyo Roshi, Tokyo, Japan) was used as a virus adsorbent, and the viruses adsorbed on the membrane were eluted into
3% beef extract solution (approximately 10 to 20 ml) by sonic treatment (type K-8814; Kontes). The concentrates thus obtained were
inoculated into a total of 30 tube cultures of Vero, RD18S, MA104, and
monkey kidney cells. Virus multiplication was checked by cytopathic
effect. Isolates were identified by a neutralization test with
poliovirus type-specific polyclonal antisera (25 U, rabbit sera). The
neutralization test was performed according to standard procedures
(24). The antisera were prepared by immunizing rabbits with
Sabin type 1, 2, or 3 strains (Denka-Seiken, Tokyo, Japan).
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FIG. 1.
Distribution of poliovirus strains isolated from river
water and sewage samples. Each dot indicates one poliovirus strain.
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A total of 78 poliovirus strains were isolated from the samples from
stations I, S, O, and G. Of these isolates, 6 (3 of type
2 and 3 of
type 3) were isolated from the river water and 72 (16
of type 1, 28 of
type 2, and 28 of type 3) were from the sewage
samples (Fig.
1). OPV
vaccination for children has been routinely
given in May and October
every year in Toyama Prefecture. Most
isolates were found during the
period following vaccination (Fig.
2). It
has been reported that most excretion periods for poliovirus
after OPV
immunization of children are from 1 to 2 months (
1,
23). In
our study, the isolates had circulated in the environmental
water for
up to 3 months after OPV immunization. Though the excretion
periods of
type 2 and 3 strains were considered to be longer than
that of type 1 (
19), the differences in excretion periods among
these three
types of isolates were not observed.
Reference polioviruses.
The isolates were compared with the
following strains: Sabin (types 1, 2, and 3), Mahoney (type 1), Lansing
(type 2), Saukett (type 3), and polio vaccine viruses (F115, type 1;
F209, type 2; F313, type 3) used in Japan during 1993 to 1995. Sabin
and wild-type strains were obtained from the National Institute of Infectious Diseases, Tokyo, Japan. Vaccine viruses of Japan were prepared in the Japan Poliomyelitis Research Institute, Tokyo, Japan.
PCR-restriction fragment length polymorphism (PCR-RFLP)
analysis.
RNA of poliovirus was extracted by treatment with RNAzol
B (Tel-Test, Inc., Friendswood, Tex.) and phenol-chloroform. The cDNA
of the viral RNA was synthesized with reverse transcriptase (Perkin-Elmer, Foster City, Calif.) at 37°C for 90 min and then amplified by PCR. The PCR was performed on a DNA thermal cycler (PJ2000; Perkin-Elmer) with 35 cycles of denaturation (94°C, 30 s), annealing (45°C, 1 min), and elongation (72°C, 1 min). The downstream primer (UC1) had the sequence
5'-GAATTCCATGTCAAATCTAGA-3', and the upstream primer (UG1)
had the sequence 5'-TTTGTGTCAGCGTGTAATGA-3' (2).
The amplified fragments (474 to 480 bp) were then separately digested
with the restriction enzyme DdeI (5 U), HpaII (14 U), or HaeIII (10 U) (all from Pharmacia Biotech) at 37°C
for 2 h. Digested products were electrophoresed on a 3.5% agarose
gel, stained with ethidium bromide, and visualized by UV illumination.
According to the results of PCR-RFLP analysis for all isolates, 71 had
restriction patterns similar to those of the homotypic
Sabin strains
and 7 had different patterns. Among type 1 isolates,
strains G4-2,
G4-12, G26-11, and G28-3 had restriction patterns
different from that
of Sabin 1 with
HaeIII (Fig.
3A). Among those
of type 2, strains O41-1
and G27-14 had patterns different from
those of Sabin 2 with
DdeI and
HaeIII, respectively (Fig.
3B).
The type 3 isolate, strain G19-4, had a pattern different from
that of
Sabin 3 with
DdeI, and the patterns obtained with
three
restriction enzymes were similar to those of the Saukett strain
(Fig.
3C). RFLP patterns different from those of Sabin strains
were
confirmed by subsequent sequence analysis of those strains
(Table
1). The RFLP patterns of vaccine strains
used in Japan
were similar to those of Sabin strains.
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FIG. 3.
RFLP patterns of the reference strains and isolates.
Reference strains: Sabin 1, 2, and 3, Mahoney, Lansing, and Saukett.
Isolates: G17-21, G4-2, G3-1, O41-1, G27-14, G4-18, and G19-4. UC,
uncut; MW, DNA molecular weight marker (MspI digest of
pBR322). Strains G17-21, G3-1, and G4-18 had restriction patterns
similar to those of Sabin strains. G4-2, O41-1, G27-14, and G19-4 had
patterns different from those of Sabin strains.
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|
Sequence analysis.
An appropriate DNA-sized band of PCR
product was extracted from a 1.5% SeaKem GTG agarose gel (FMC
Bioproducts, Rockland, Maine) and purified by passage through SUPREC-01
(TaKaRa Shuzo Co., Ltd., Shiga, Japan). The purified PCR product was
sequenced by using a dRhodamine Terminator cycle sequencing
reaction with a DNA sequencing kit (PE Applied Biosystems) and a
genetic analyzer (ABI Prism 310; Perkin-Elmer). The sequence data (type
1, positions 2422 to 2860; type 2, 2424 to 2862; type 3, 2419 to 2851)
on the VP3 and VP1 regions were aligned by using the software Gene
Works version 2.3.1 (IntelliGenetics Inc.), and amino acid sequences were deduced.
Of 78 isolates, the nucleotide sequences of 36 isolates (4 of type 1, 17 of type 2, and 15 of type 3) were identical to those
of homotypic
Sabin strains. The other 42 isolates (12 of type
1, 14 of type 2, and
16 of type 3) were Sabin variants that had
less than 1.4% nucleotide
divergence from homotypic Sabin in the
VP3 and VP1 region (Table
1). On
the other hand, the nucleotide
sequences of vaccine viruses used in
Japan were identical to those
of homotypic Sabin strains. Therefore, it
is suggested that the
mutations in the variant isolates occurred after
administration
of the vaccine. The mutation of the poliovirus genome
during replication
in human intestine or in vitro has been reported
previously (
7,
8,
9,
12,
16). Thirty-two of these variants
had one
to three amino acid
substitutions.
Among the 12 type 1 variants, mutations of one to six nucleotides per
strain were found. Strain G28-3 had four nucleotide
mutations, 3 (at
positions 2545, 2749, and 2795) for which there
was reversion toward
the Mahoney strain. Nine variants had mutation
A
G at nucleotide
position 2795, which was a reversion toward
the Mahoney strain.
According to Bouchard et al. (
3), the mutation
in this
position has been related to attenuation. It should be
noted that this
back mutation has occurred in many isolates. Moreover,
the mutation
induced the amino acid substitution Thr
106Ala in
VP1,
which is located near the neutralization antigenic site (N-Ag
1). Other
mutations close to N-Ag 1 were found at amino acid position
90 (Ile
Met) in strain G28-3 and at position 99 (Lys
Glu) in G3-11
and
in G28-9 (Lys
Asn). In order to clarify the effect of amino
acid
substitution close to N-Ag 1, the neutralization titers of
type-specific polyclonal antibody against type 1 isolates were
assayed
(Table
2). Sabin variants were easily
neutralized at
approximately the titer for the Sabin type 1 strain by
type 1
antibody but not by type 2 and 3 antibodies. This suggests that
OPV immunization might give protective immunity against infection
with vaccine variants. Among 14 type 2 variants, mutations of
one to
three nucleotides per strain were found at random positions.
No
mutation near N-Ag 1 was found. Among 16 type 3 variants, mutations
of
one to four nucleotides per strain were found. A mutation close
to N-Ag
1 was found at amino acid position 105 (Met
Thr) in strains
G4-18,
G5-1, and G27-3. These three variants were neutralized
at approximately
the same titer as the Sabin type 3 strain by
type 3 antibody (data not
shown).
Concluding remarks.
The present study shows that
poliovirus isolates were all derived from the Sabin vaccine
strains, suggesting that Toyama Prefecture, Japan, is a
wild-poliovirus-free area.
It has been reported that RFLP analysis was useful for intratypic
differentiation between wild and vaccine strains (
2,
10,
25). Seven of 78 isolates were differentiated as non-Sabin-like
strains by RFLP analysis. On the other hand, sequencing analysis
showed
that 42 of those isolates were Sabin variants that had
less than 1.4%
nucleotide divergence from Sabin strains, including
the 7 isolates
differentiable by RFLP patterns. Results of RFLP
and sequencing
analyses revealed little difference in identifying
identical and
mutated isolates between the two methods. Variants
were found in types
1, 2, and 3 after OPV immunization. Though
the RFLP analysis is useful
for intratypic differentiation among
isolates, it should be applied
more carefully to vaccine variants
appearing immediately after OPV
immunization of
children.
Though the sewage system served an average of 50% of the population in
Toyama Prefecture during 1993 to 1995, sanitary conditions
were
supposed to be relatively good because of improved domestic
drainage
disposal and lifestyle, including the use of paper diapers
in baby
care. Of 78 isolates, 6 were isolated from river water
samples and the
rest were from sewage samples. Therefore, it is
suggested that the most
polioviruses from humans had accumulated
in sewage. Because of the
difficulty of giving the general population
access to the sewage
system, the risk of infection with vaccine
variants in the environment
may be considered low. However, it
will be necessary to assay the
possible neurovirulence of vaccine
variants from the environment.
Since the year 2000, an inactivated
polio vaccine
immunization program in the United States has started
to reduce the
risk of infection by revertants excreted from humans
(
6).
Likewise, in terms of infection from environmental isolates,
the
introduction of an inactivated polio vaccine would contribute
to the
final stage of the polio eradication
program.
| |
ACKNOWLEDGMENTS |
We are grateful to T. Yoneyama and A. Hagiwara (National Institute
of Infectious Diseases) for distribution of the polioviruses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Toyama
Institute of Health, Nakataikoyama, Kosugi-machi, Imizu-gun,
Toyama 939-0363, Japan. Phone: 81-766-56-5506. Fax: 81-766-56-7326. E-mail: Kumiko.matsuura{at}tak.pref.toyama.jp.
| |
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Applied and Environmental Microbiology, November 2000, p. 5087-5091, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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