AEM Accepted Manuscript Posted Online 2 January 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.03575-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Environmental Surveillance of Poliovirus in Sewage Water around the Introduction Period of
2
Inactivated Polio Vaccine in Japan
3 4 5 6
Tomofumi Nakamura1, 2, Mitsuhiro Hamasaki2, Hideaki Yoshitomi2, Tetsuya Ishibashi2, Chiharu
7
Yoshiyama2, Eriko Maeda2, Nobuyuki Sera2*, Hiromu Yoshida1
8 9 10 11
1
12
Musashimurayama-shi, Tokyo 208-0011, Japan
13
2
14
818-0135, Japan
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen,
Fukuoka Institute of Health and Environmental Sciences, Mukaizano 39, Dazaifu-shi, Fukuoka
15 16 17 18 19 20 21 22 23 24 25 26 27
*Correspondence should be addressed to:
28
Nobuyuki Sera
29
Fukuoka Institute of Health and Environmental Sciences, Mukaizano 39, Dazaifu-shi, Fukuoka
30
818-0135, Japan
31
Tel:+81-92-921-9945
32
Fax:+81-92-928-1203
33
E-mail: [email protected]
1
34
ABSTRACT
35
Environmental virus surveillance was conducted at two independent sewage plants from urban and
36
rural areas in the northern prefecture of the Kyushu district, Japan, to trace the polioviruses (PVs)
37
within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to
38
January 2013. The frequency of PV isolation at the urban plant was 1.5-times higher than that at the
39
rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the
40
PV isolates the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited
41
more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated
42
poliovirus vaccine (IPV) was introduced for routine immunization on September 1, 2012, replacing
43
the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage
44
waters declined before OPV cessation at both sites. Our study highlights the importance of
45
environmental surveillance to detect the excretion of PVs from an OPV-immunized population in a
46
highly sensitive manner, during the OPV to IPV transition period.
47 48
(170 words)
2
49
INTRODUCTION
50
Poliovirus (PV) is a non-enveloped, positive-sense single-stranded RNA virus belonging to the genus
51
Enterovirus of the family Picornaviridae (1, 2). PV possesses a relatively small icosahedral particle
52
structure (approximately 30 nm in diameter) composed of four different capsid proteins, including
53
VP1, where most antigenic epitopes locate (3, 4). Similar to other non-polio enteroviruses (NPEVs),
54
PV is transmitted via the fecal–oral route and efficiently replicates in the intestinal tract (3). During
55
PV infection, the virus is excreted from the human gut into the stool for approximately 2 months
56
(5-7). Although most PV infections are asymptomatic, patients can develop poliomyelitis following
57
viremia in some cases, resulting in residual paralysis (8).
58
Since the live oral poliovirus vaccine (OPV) was introduced in many industrial countries in the
59
1960s, polio epidemics have been successfully controlled. In 1988, the World Health Assembly
60
resolved to eradicate polio by launching the Global Polio Eradication Initiative (GPEI). The
61
large-scale OPV immunization resulted in a drastic reduction in the number of poliomyelitis cases. To
62
date,
63
(http://www.polioeradication.org/Dataandmonitoring.aspx) and the WHO is closely monitoring the
64
neighboring countries at increased risk of re-emergence of wild or vaccine-derived poliovirus (VDPV)
65
to maintain a polio-free situation. In Japan, the last indigenous wild PV was isolated from a single
66
patient with poliomyelitis in 1980 (9). In an effort to remain polio-free, OPV has been used for routine
67
immunization for the last 50 years in Japan. It was scheduled twice for children between 3- to
68
18-months-old at the interval of more than 6 weeks, and immunized mainly in the spring and autumn
69
season at school and hospital. To minimize the risk of vaccine-associated paralytic poliomyelitis
70
(VAPP) due to OPV, standalone conventional inactivated poliovirus vaccine (cIPV) was introduced in
71
September 2012. Thereafter Sabin-derived IPV in combination with Diphtheria, Tetanus, and
the
only
polio
endemic
countries
3
are
Nigeria,
Pakistan,
and
Afghanistan
72
Pertussis vaccine (DTP-sIPV) (10) was introduced in November 2012 for routine immunization (three
73
doses administered from 3- to 12-months-old babies, with one booster dose the age of 12 and 15
74
months after the 3rd immunization). Alarmingly, the OPV national coverage declined to 67.2% at the
75
immunization period of spring 2012, prior to the transition to IPV (11). The refusal of OPV
76
immunization was most likely due to the growing public concern about VAPP (12, 13). Consequently,
77
the risk of PV infection has increased among unvaccinated children and in the larger population.
78
Environmental surveillance is a highly sensitive method for detecting enteroviruses such as PVs
79
in environmental samples, and the practice has been adopted by many countries and regions
80
worldwide (14-20). It is critically important to routinely monitor sentinel sites for the emergence of
81
novel VDPV strains and the importation of wild PV from endemic countries. In the global effort to
82
eradicate polio, IPV immunization will be introduced before trivalent OPV cessation to minimize the
83
risk of VAPP spread to susceptible individuals within the population (21). We recently designed a
84
comprehensive monitoring system for the surveillance of enteric viruses at sentinel hospitals (22) and
85
to determine the relationship between environmental and patient surveillance. This study led to the
86
isolation of enteroviruses and PVs. Here we report the prevalence of PVs in sewage water from two
87
locations in Japan during the OPV to IPV transition period. Our results provide valuable information,
88
at the local community level, on the impact of the transition period of PV immunization with
89
considerations on how OPV can be safely discontinued at the global level.
4
90
MATERIALS AND METHODS
91
Sample Collection
92
Influent wastewater was obtained from two sewage disposal plants (T and Y) located in the northern
93
area of Kyushu, Japan. The sanitation coverage in the area, defined as the percentage of the
94
population connected to the sewage system relative to the entire population, is 61% (23). The T plant
95
is located in an urban area and has high sanitation coverage (≥90%). The Y plant is located in a rural
96
area and has low sanitation coverage (≤10%). The watershed population was similar between the two
97
areas. Approximately 190,000 persons live around the T plant, and approximately 180,000 persons
98
live around the Y plant. The child population of recommended age for OPV immunization (0–2 years
99
old) was approximately 2,300 children in the T plant watershed population and 1,400 in the Y plant
100
(Table 1). Every first week of the month, 1,000 ml of wastewater was routinely obtained at each plant
101
and stored at 4°C until further processing.
102 103
Treatment of the Wastewater Samples
104
The samples were concentrated as described previously (15, 24, 25). In brief, the collected wastewater
105
was centrifuged at 1,500 × g (30 min; 4°C). Following this, MgCl2 was added to the resulting
106
supernatant to a final concentration of 0.05 M, and the solution was adjusted to pH 3.5 with HCl. The
107 108
filter, Advantec, Tokyo, Japan). The filter holder was equipped with a PST-1000 digital tube pump
109
(Iwaki, Tokyo, Japan). The membrane was ground in 10 ml of 3% beef extract with a MediFASTH2
110
homogenizer (Omni International) to elute the bound viruses. After membrane grinding, the elution
111
was centrifuged at 16,000 × g for 30 min to remove debris and filtered with a 0.22- or
112
0.45-
-pore-size PVDF membrane filter (Millipore). The final concentrates were stored at −20°C
5
113
until further analysis.
114 115
Virus Isolation
116
The procedure involved two cultivation steps. First, 100 l for each concentrate was inoculated into
117
four wells of five cultured cell lines (Vero-E6, LLC-MK2, HEp-2, FL, and RD-18S) grown and
118
maintained in 24-well plates containing Dulbecco’s Modified Eagle’s Medium (MEM medium,
119
Sigma-Aldrich). Three cell lines (LLC-MK2, HEp-2, and FL) were purchased from Dainippon
120
Pharmaceutical (Japan). Each cell line has a different range of sensitivity for virus isolation, and the
121
RD-18S cell line was previously described as advantageous for the isolation of coxsackievirus A (26).
122
The cell lines were observed for 7 days. And samples with no cytopathic effect (CPE) were passed to
123
new well plate and observed for another 7 days. During the observation period, the viruses isolated
124
from the cell culture showed a CPE. Second, the supernatant of each CPE positive culture fluids (100
125
L) onto L20B cells maintained in MEM medium for selectively isolating PVs and further studying
126
CPE for 7 days (27, 28). For the screening test of non-poilo enteroviruses (NPEVs), reverse
127
transcription (RT), conventional PCR and direct sequencing were carried out against all CPE-positive
128
agents as previously described (2X).
129 130
Serotype Identification and VP1 sequencing of PVs
131
For CPE positive culture fluids through the L20B cell inoculation, we performed neutralization test
132
(NT) with poliovirus type-specific antisera (DENKA SEIKEN, Tokyo, Japan), in order to isolate each
133
serotype from poliovirus mixtures (15).
134
Total viral RNA was extracted from poliovirus isolate, using the QIAcube Automated DNA/RNA
135
Purification System (Qiagen, Tokyo, Japan) on the basis of the QIAamp Viral RNA Mini Kit
6
136
procedure (Qiagen), according to the manufacturer’s instruction. Reverse transcription polymerase
137
chain reaction (RT-PCR) was performed on the VP1 region of the viral genome using the UG1/UC11
138
specific primer set for PV (30) and the One Step RT-PCR Kit (Qiagen). Direct sequencing was
139
performed for samples with effective amplification confirmed by a single and specific gel band by
140
electrophoresis. The PCR amplicons were enzymatically purified with Illustra ExoStar (GE
141
Healthcare, Tokyo, Japan). The sequencing reactions were conducted using the BigDye Terminator
142
v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan). The sequenced products were
143
purified using the BigDye XTerminator Purification Kit (Applied Biosystems) and sequenced with the
144
3130xl Genetic Analyzer (Applied Biosystems). The full-length VP1 sequence of PV was aligned
145
using MEGA5 software (31). The VP1 sequences of the reference vaccine strains used in this study
146
were Sabin 1 (AY082688, 906 bp), Sabin 2 (AY082679, 903 bp), and Sabin 3 (AY082683, 900 bp).
147
The entire VP1 nucleotide sequence of the 83 PV isolates in this study was deposited in the GenBank
148
database under the accession numbers AB829440–AB829449, AB829551, AB829553–AB829562,
149
AB829564–AB829572, AB829574–AB829600, AB921169–AB921180, and AB980981–AB980994.
7
150
RESULTS
151
Isolation of PVs from sewage water
152
Concentrates from the wastewater collected at the two sewage plants in the northern prefecture of the
153
Kyushu district, Japan, were inoculated onto five different cell lines. During the 34-month sampling
154
period, 446 CPE-positive cultures were identified using these 5 cell lines. After further reinoculation
155
of all CPE-positive samples into L20B cultures, we confirmed CPE in 103 cultures in total. As a result
156
of following NT, we isolated 83 PVs and 22 non-polio viruses (Table 1).
157
The seasonal distribution of PVs and NPEVs during the study is presented in Figure 1. After
158
re-isolation of a single PV serotype by the NT assay, we conducted sequence analysis of the PV
159
capsid VP1 gene. The serotype frequency of PV-positive isolates was 23 type 1 (PV1), 38 type 2
160
(PV2), and 22 type 3 (PV3) (Table 1). Sequence analysis of the full-length VP1 region showed that all
161
isolates were Sabin-like, with 1% nucleotide divergence from Sabin 1 and Sabin 3 and 0.6%
162
divergence from Sabin 2 (32). As shown in Table 1, the frequency of PV-positive isolates obtained at
163
the T plant was >1.5-times higher than that of isolates obtained at the Y plant. The distribution of each
164
serotype showed a similar trend, with a greater frequency at the T plant than at the Y plant. These
165
distributions mirrored the municipal vaccination (OPV) period in each area for approximately 2–3
166
months (Fig. 1). The frequency of isolated PVs was highest in 2010, and tended to gradually decrease
167
in the successive months of the sampling period. The last isolation of PV was in May 2012 at the T
168
plant and November 2011 at the Y plant. Since September 2012, routine IPV immunization is
169
conducted nationwide. Thus, before the immunization transition, PV originating from OPV had
170
disappeared from sewage water at both plants. On the other hand, NPEVs have been isolated over
171
research period (data not shown).
172
8
173
Nucleotides substitutions on VP1 region
174
The use of primers specific to the VP1 region of each PV serotype revealed mutations in the viral
175
capsid sequence of the PV isolates, relative to the Sabin vaccine strains. Of the 83 PV isolates
176
obtained during this study, 69 (83%) showed at least one mutation; two PV3 variants had six
177
nucleotide mutations in VP1 (Fig. 2).
178
With regard to the mutations detected in the VP1 region, we closely examined the
179
nonsynonymous mutations affecting the codons of amino acid residues. Many of these mutations are
180
“attenuation markers” responsible for the attenuation phenotype of OPV strains (4). Several groups
181
reported the following markers in the VP1 region of the Sabin vaccine strains: T106 and F134 in Sabin 1
182
(S1) (33), I143 in Sabin 2 (S2) (34), and T6 in Sabin 3 (S3) (35, 36). In the VP1 sequence of the 23 S1
183
isolates, we found 12 isolates with an amino acid substitution at T106 (52%) (Fig. 3). All these
184
mutations exhibited nonsynonymous mutations T → A/S at residue 106 from the reference S1 strain.
185
In contrast, no modified codon was found in the F134-coding region of the S1 isolates. Among the 38
186
S2 isolates, we found 20 isolates with the mutation at the I143 residue (53 %). These mutations also
187
included three types of nonsynonymous substitutions (I143 → T/V/N) affected by the first and second
188
nucleotide modifications of this codon. On the other hand, OPV Sabin 3 strain has been known to
189
contain mutations at the second codon of the 6th amino acid position of VP1 even in the
190
manufacturing. In fact, all PV3 isolates had isoleucine at the 6th position unlike original Sabin 3
191
having threonine. In this study, we described the amino acid change as mutation. In summary,
192
molecular analysis of the isolated PV serotypes revealed that several isolates contained mutations
193
affecting amino acid residues of known “attenuation markers” of VP1.
9
194
DISCUSSION
195
Environmental surveillance of PVs is critical to maintain polio-free areas and to work toward the
196
global eradication of polio. We present the results of a 34-month environmental virus surveillance
197
study, conducted at two independent sewage disposal plants located in urban and rural areas of
198
northern Kyushu district, Japan. In total, 83 Sabin-like PVs were isolated from wastewater samples
199
collected monthly from April 2010 to January 2013. The surrounding areas of each plant had unique
200
features, including differences in sewage line coverage and infant age distribution, despite the similar
201
population size (Table 1). We sought to determine the frequency of PV isolation from wastewater
202
around the OPV to IPV transition period in those communities.
203
In Japan, the usage rate of disposable diapers is quite high (approximately 80%–90%). If
204
disposable diapers containing stool from OPV-immunized children are properly treated and discarded,
205
the frequency of PV detection in sewage water would presumably be quite low. However, PV was
206
detected in wastewater sampled from two areas (T and Y disposal plants) with scheduled OPV
207
immunizations and different infant age distributions. This finding suggests that either stool from
208
OPV-immunized children flowed into the sewage line and contaminated the water or intrafamilial
209
OPV transmission led to PV excretion into the sewage water. In fact, several reports have indicated
210
intrafamilial and interfamilial spread of PV, including the vaccine strain and NPEV (37-39).
211
Because of the high usage rate of disposable diapers, PVs from OPV-immunized children may be
212
transmitted by familial contacts, such as siblings and parents, or contacts at school facilities, such as
213
kindergarteners (40). However, we would also anticipate NPEV to be widely transmitted among
214
family members. In Toyama, Japan, a correlation was established between the high frequency of
215
echovirus type 13 (E13) isolation from environmental water in the summer and seroconversion
216
against E13 between preoutbreaks and postoutbreaks, regardless of age (41). These studies suggest
10
217
that we should expect differences in the frequency of NPEV between the two plants. On the other
218
hand, we showed that the frequency of PV was clearly related to the OPV vaccination period.
219
Previous studies have reported a rapid decline in PV isolation around the OPV to IPV transition
220
period, with a disappearance of PV vaccine strains from wastewater within 2 to 3 months after the
221
cessation of OPV administration (42-45). In contrast, our study showed the disappearance of
222
Sabin-like PVs from the environment before OPV immunization had ceased (Fig. 1). PVs have not
223
been detected from wastewater at the T and Y plant since June 2012 and December 2011, respectively.
224
After the announcement that IPV would replace OPV for routine immunization in Japan (May 2011),
225
the nationwide coverage by routine OPV immunization declined until IPV was introduced in
226
September 2012, mostly because of public concerns about VAPP (12, 13). The rate of VAPP is
227
assuming 1 per two to five million inoculations in Japan. However, the rate might not be low for the
228
parents have children should be received polio vaccine in the period. And from now on, it will be very
229
important to monitor the decline and disappearance of VAPP cases after OPV cessation, as
230
demonstrated in USA (46).
231
The analysis of the viral capsid VP1 sequence showed that all PV isolates were Sabin-like
232
vaccine strains. Many isolates contained mutations in the VP1 sequence. However, major deviations
233
from the attenuated Sabin strains were relatively rare, and there was no significant difference in
234
mutations between the virus serotypes. Importantly, these data show that no VDPV has emerged and
235
circulated in the sampling sites over the course of this study.
236
The PV1 isolates also presented several mutations affecting codons and amino acid residues,
237
known as attenuation markers, in the VP1 region of all PV serotypes. In the Sabin 1-like isolates, the
238
T → A amino acid mutation at position 106 was the most frequent. This mutation introduces a residue
239
found in the Mahoney strain, a neurovirulent parent of the Sabin 1 strain. With regard to the Sabin
11
240
2-like isolates, the I143 position was mutated (I → T/V/N) in 53% of the isolates. All Sabin 3-like
241
strains isolated in this study had isoleucine at the 6th position of VP1 amino acid unlike the original
242
Sabin 3 strain having threonine. It is well known that Sabin 3 of OPV originally has mixed
243
nucleotides at second codon of the relevant position even in manufacturing (47). But it is difficult for
244
us to know at what stage the mutations occurred. And our study have not intended to show the
245
virulence and replication ability of isolates but just the numbers of VP1 mutation for denying the
246
emergence of VDPV in the research field. This is one of the limitation points of this study. Besides
247
this, the strains have isolated from sewage water not from patients and other healthy persons. That’s
248
why we can only speculate when the mutations occurred and the effect of VP1 mutations to virulence
249
of the isolates. More information may be obtained when we sequence genome of the isolates and
250
compare it with known marker of other regions. However, our results indicate that the continuous
251
surveillance of nucleotide substitutions is necessary to monitor the emergence of VDPVs and other
252
mutation strains.
253
In conclusion, we offer an environmental surveillance strategy that can detect the excretion of
254
PVs from OPV-immunized populations with high sensitivity. Needless to say, the transition from OPV
255
to IPV is an essential step toward the global eradication of PV. However, the transition from OPV to
256
IPV should be carefully orchestrated before OPV cessation to maintain immunity coverage by OPV.
257
Even if the IPV transition is successful, the risk of PV infection for a susceptible population must be
258
closely monitored with a high-quality surveillance system because PV can be silently transmitted and
259
its viral genome can mutate during replication in the human gut, partly because of insufficient
260
mucosal immunity (48, 49). Under these circumstances, the environmental surveillance of PV plays a
261
key role not only to monitor the importation of wild-type PV from endemic areas but also to prepare
262
against emerging VDPVs before the global cessation of OPV at the final stage of polio eradication.
12
263 264
Acknowledgement
265
This study was supported by a grant for Research on Emerging and Re-emerging Infectious
266
Diseases from the Ministry of Health, Labour and Welfare of Japan. We appreciate the technical
267
suggestions of Drs Takenori Takizawa and Masae Iwai-Itamochi from the Toyama Institute of Health
268
and the critical comments of Drs Hiroyuki Shimizu and Chikako Kataoka from the National Institute
269
of Infectious Diseases, Japan.
13
270
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M, Daikoku T, Shiraki K. 2010. Widespread circulation of echovirus type 13 demonstrated by
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Fitzsimons A, Garrett N, Graham D, Lennon D, Shimizu H, Miyamura T, Pallansch MA. 2005.
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43. Mueller JE, Bessaud M, Huang QS, Martinez LC, Barril PA, Morel V, Balanant J, Bocacao
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PM, Cono J, Wharton M, Orenstein WA, Sutter RW. 2004. Vaccine policy changes and
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epidemiology of poliomyelitis in the United States. JAMA. 292:1696-1701.
445 446
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447
Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral
448
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Shulman LM, Mendelson E, Gdalevich M, Lev B, Gamzu R, Grotto I. 2013. Insidious
452
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453 454
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456
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457
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19
458
Figure Legends
459 460
Figure 1. Frequency of poliovirus (PV) and non-polio enterovirus (NPEV) isolation at two
461
independent sewage plants
462
Plants T and Y were compared for the frequency and distribution of NPEV and poliovirus type 1, type
463
2, and type 3 isolated over the 34-month collection period. The double-headed arrows above each
464
graph indicate scheduled vaccination periods in each area. The dotted vertical line indicates the month
465
and year when IPV was introduced in Japan. The first Y axis (left) indicates the frequency of PV
466
isolation each month, and the second Y axis (right) indicates the frequency of NPEV isolation each
467
month. Sample collection was conducted from April 2010 to January 2013 (X axis).
468 469
Figure 2. Distribution of nucleotide substitutions in the VP1 regions of the isolated strains of
470
polioviruses (PV)
471
The nucleotide divergence in VP1 between isolates and reference vaccine strains (Sabin 1: AY082688,
472
Sabin 2: AY082679, and Sabin 3: AY082683) is shown on the X axis. The number of isolates with
473
each mutation is shown on the Y axis.
474 475
Figure 3. Nucleotides substitutions on VP1 region
476
Above each nucleotide, the column indicates the percentage of all PVs of an isolated serotype during
477
the collection period that featured a mutation at this position. Below each codon, the circle shows the
478
amino acid substitution resulting from the mutation. The letters below the nucleotides and capital in
479
the circles represent conventional abbreviations for amino acids. *The OPV Sabin 3 originally
480
contains mutation at the second codon of VP1 6th amino acid known as “mix base position”.
20
Table 1
Table 1 Comparison of two sewage plants appearance and isolated serotype of poliovirus
Isolated serotyope (%)a
Environment
Watershed population
0-2 years old populaton
Sanitation Coverage
CPE-positive
L20B CPEpositive
PV isolates (NT)
PV1
PV2
PV3
T plant
urban
190,000
2,300
≥ 90%
248
67
52
16 (31)
25 (48)
11 (21)
Y plant
rural
180,000
1,400
≤ 10%
198
36
31
7 (23)
13 (42)
11 (35)
Total
-
-
-
-
446
103
83
23 (28)
38 (46)
22 (27)
a) % are calculated from the numbers of all isolated PVs of the area
1
Environmental Surveillance of Poliovirus in Sewage Water around the Introduction Period of
2
Inactivated Polio Vaccine in Japan
3 4 5 6
Tomofumi Nakamura1, 2, Mitsuhiro Hamasaki2, Hideaki Yoshitomi2, Tetsuya Ishibashi2, Chiharu
7
Yoshiyama2, Eriko Maeda2, Nobuyuki Sera2*, Hiromu Yoshida1
8 9 10 11
1
12
Musashimurayama-shi, Tokyo 208-0011, Japan
13
2
14
818-0135, Japan
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen,
Fukuoka Institute of Health and Environmental Sciences, Mukaizano 39, Dazaifu-shi, Fukuoka
15 16 17 18 19 20 21 22 23 24 25 26 27
*Correspondence should be addressed to:
28
Nobuyuki Sera
29
Fukuoka Institute of Health and Environmental Sciences, Mukaizano 39, Dazaifu-shi, Fukuoka
30
818-0135, Japan
31
Tel:+81-92-921-9945
32
Fax:+81-92-928-1203
33
E-mail: [email protected]
1
34
ABSTRACT
35
Environmental virus surveillance was conducted at two independent sewage plants from urban and
36
rural areas in the northern prefecture of the Kyushu district, Japan, to trace the polioviruses (PVs)
37
within communities. Consequently, 83 PVs were isolated over a 34-month period from April 2010 to
38
January 2013. The frequency of PV isolation at the urban plant was 1.5-times higher than that at the
39
rural plant. Molecular sequence analysis of the viral VP1 gene identified all three serotypes among the
40
PV isolates the most prevalent serotype being type 2 (46%). Nearly all poliovirus isolates exhibited
41
more than one nucleotide mutation from the Sabin vaccine strains. During this study, inactivated
42
poliovirus vaccine (IPV) was introduced for routine immunization on September 1, 2012, replacing
43
the live oral poliovirus vaccine (OPV). Interestingly, the frequency of PV isolation from sewage
44
waters declined before OPV cessation at both sites. Our study highlights the importance of
45
environmental surveillance to detect the excretion of PVs from an OPV-immunized population in a
46
highly sensitive manner, during the OPV to IPV transition period.
47 48
(170 words)
2
49
INTRODUCTION
50
Poliovirus (PV) is a non-enveloped, positive-sense single-stranded RNA virus belonging to the genus
51
Enterovirus of the family Picornaviridae (1, 2). PV possesses a relatively small icosahedral particle
52
structure (approximately 30 nm in diameter) composed of four different capsid proteins, including
53
VP1, where most antigenic epitopes locate (3, 4). Similar to other non-polio enteroviruses (NPEVs),
54
PV is transmitted via the fecal–oral route and efficiently replicates in the intestinal tract (3). During
55
PV infection, the virus is excreted from the human gut into the stool for approximately 2 months
56
(5-7). Although most PV infections are asymptomatic, patients can develop poliomyelitis following
57
viremia in some cases, resulting in residual paralysis (8).
58
Since the live oral poliovirus vaccine (OPV) was introduced in many industrial countries in the
59
1960s, polio epidemics have been successfully controlled. In 1988, the World Health Assembly
60
resolved to eradicate polio by launching the Global Polio Eradication Initiative (GPEI). The
61
large-scale OPV immunization resulted in a drastic reduction in the number of poliomyelitis cases. To
62
date,
63
(http://www.polioeradication.org/Dataandmonitoring.aspx) and the WHO is closely monitoring the
64
neighboring countries at increased risk of re-emergence of wild or vaccine-derived poliovirus (VDPV)
65
to maintain a polio-free situation. In Japan, the last indigenous wild PV was isolated from a single
66
patient with poliomyelitis in 1980 (9). In an effort to remain polio-free, OPV has been used for routine
67
immunization for the last 50 years in Japan. It was scheduled twice for children between 3- to
68
18-months-old at the interval of more than 6 weeks, and immunized mainly in the spring and autumn
69
season at school and hospital. To minimize the risk of vaccine-associated paralytic poliomyelitis
70
(VAPP) due to OPV, standalone conventional inactivated poliovirus vaccine (cIPV) was introduced in
71
September 2012. Thereafter Sabin-derived IPV in combination with Diphtheria, Tetanus, and
the
only
polio
endemic
countries
3
are
Nigeria,
Pakistan,
and
Afghanistan
72
Pertussis vaccine (DTP-sIPV) (10) was introduced in November 2012 for routine immunization (three
73
doses administered from 3- to 12-months-old babies, with one booster dose the age of 12 and 15
74
months after the 3rd immunization). Alarmingly, the OPV national coverage declined to 67.2% at the
75
immunization period of spring 2012, prior to the transition to IPV (11). The refusal of OPV
76
immunization was most likely due to the growing public concern about VAPP (12, 13). Consequently,
77
the risk of PV infection has increased among unvaccinated children and in the larger population.
78
Environmental surveillance is a highly sensitive method for detecting enteroviruses such as PVs
79
in environmental samples, and the practice has been adopted by many countries and regions
80
worldwide (14-20). It is critically important to routinely monitor sentinel sites for the emergence of
81
novel VDPV strains and the importation of wild PV from endemic countries. In the global effort to
82
eradicate polio, IPV immunization will be introduced before trivalent OPV cessation to minimize the
83
risk of VAPP spread to susceptible individuals within the population (21). We recently designed a
84
comprehensive monitoring system for the surveillance of enteric viruses at sentinel hospitals (22) and
85
to determine the relationship between environmental and patient surveillance. This study led to the
86
isolation of enteroviruses and PVs. Here we report the prevalence of PVs in sewage water from two
87
locations in Japan during the OPV to IPV transition period. Our results provide valuable information,
88
at the local community level, on the impact of the transition period of PV immunization with
89
considerations on how OPV can be safely discontinued at the global level.
4
90
MATERIALS AND METHODS
91
Sample Collection
92
Influent wastewater was obtained from two sewage disposal plants (T and Y) located in the northern
93
area of Kyushu, Japan. The sanitation coverage in the area, defined as the percentage of the
94
population connected to the sewage system relative to the entire population, is 61% (23). The T plant
95
is located in an urban area and has high sanitation coverage (≥90%). The Y plant is located in a rural
96
area and has low sanitation coverage (≤10%). The watershed population was similar between the two
97
areas. Approximately 190,000 persons live around the T plant, and approximately 180,000 persons
98
live around the Y plant. The child population of recommended age for OPV immunization (0–2 years
99
old) was approximately 2,300 children in the T plant watershed population and 1,400 in the Y plant
100
(Table 1). Every first week of the month, 1,000 ml of wastewater was routinely obtained at each plant
101
and stored at 4°C until further processing.
102 103
Treatment of the Wastewater Samples
104
The samples were concentrated as described previously (15, 24, 25). In brief, the collected wastewater
105
was centrifuged at 1,500 × g (30 min; 4°C). Following this, MgCl2 was added to the resulting
106
supernatant to a final concentration of 0.05 M, and the solution was adjusted to pH 3.5 with HCl. The
107 108
filter, Advantec, Tokyo, Japan). The filter holder was equipped with a PST-1000 digital tube pump
109
(Iwaki, Tokyo, Japan). The membrane was ground in 10 ml of 3% beef extract with a MediFASTH2
110
homogenizer (Omni International) to elute the bound viruses. After membrane grinding, the elution
111
was centrifuged at 16,000 × g for 30 min to remove debris and filtered with a 0.22- or
112
0.45-
-pore-size PVDF membrane filter (Millipore). The final concentrates were stored at −20°C
5
113
until further analysis.
114 115
Virus Isolation
116
The procedure involved two cultivation steps. First, 100 l for each concentrate was inoculated into
117
four wells of five cultured cell lines (Vero-E6, LLC-MK2, HEp-2, FL, and RD-18S) grown and
118
maintained in 24-well plates containing Dulbecco’s Modified Eagle’s Medium (MEM medium,
119
Sigma-Aldrich). Three cell lines (LLC-MK2, HEp-2, and FL) were purchased from Dainippon
120
Pharmaceutical (Japan). Each cell line has a different range of sensitivity for virus isolation, and the
121
RD-18S cell line was previously described as advantageous for the isolation of coxsackievirus A (26).
122
The cell lines were observed for 7 days. And samples with no cytopathic effect (CPE) were passed to
123
new well plate and observed for another 7 days. During the observation period, the viruses isolated
124
from the cell culture showed a CPE. Second, the supernatant of each CPE positive culture fluids (100
125
L) onto L20B cells maintained in MEM medium for selectively isolating PVs and further studying
126
CPE for 7 days (27, 28). For the screening test of non-poilo enteroviruses (NPEVs), reverse
127
transcription (RT), conventional PCR and direct sequencing were carried out against all CPE-positive
128
agents as previously described (2X).
129 130
Serotype Identification and VP1 sequencing of PVs
131
For CPE positive culture fluids through the L20B cell inoculation, we performed neutralization test
132
(NT) with poliovirus type-specific antisera (DENKA SEIKEN, Tokyo, Japan), in order to isolate each
133
serotype from poliovirus mixtures (15).
134
Total viral RNA was extracted from poliovirus isolate, using the QIAcube Automated DNA/RNA
135
Purification System (Qiagen, Tokyo, Japan) on the basis of the QIAamp Viral RNA Mini Kit
6
136
procedure (Qiagen), according to the manufacturer’s instruction. Reverse transcription polymerase
137
chain reaction (RT-PCR) was performed on the VP1 region of the viral genome using the UG1/UC11
138
specific primer set for PV (30) and the One Step RT-PCR Kit (Qiagen). Direct sequencing was
139
performed for samples with effective amplification confirmed by a single and specific gel band by
140
electrophoresis. The PCR amplicons were enzymatically purified with Illustra ExoStar (GE
141
Healthcare, Tokyo, Japan). The sequencing reactions were conducted using the BigDye Terminator
142
v3.1 Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan). The sequenced products were
143
purified using the BigDye XTerminator Purification Kit (Applied Biosystems) and sequenced with the
144
3130xl Genetic Analyzer (Applied Biosystems). The full-length VP1 sequence of PV was aligned
145
using MEGA5 software (31). The VP1 sequences of the reference vaccine strains used in this study
146
were Sabin 1 (AY082688, 906 bp), Sabin 2 (AY082679, 903 bp), and Sabin 3 (AY082683, 900 bp).
147
The entire VP1 nucleotide sequence of the 83 PV isolates in this study was deposited in the GenBank
148
database under the accession numbers AB829440–AB829449, AB829551, AB829553–AB829562,
149
AB829564–AB829572, AB829574–AB829600, AB921169–AB921180, and AB980981–AB980994.
7
150
RESULTS
151
Isolation of PVs from sewage water
152
Concentrates from the wastewater collected at the two sewage plants in the northern prefecture of the
153
Kyushu district, Japan, were inoculated onto five different cell lines. During the 34-month sampling
154
period, 446 CPE-positive cultures were identified using these 5 cell lines. After further reinoculation
155
of all CPE-positive samples into L20B cultures, we confirmed CPE in 103 cultures in total. As a result
156
of following NT, we isolated 83 PVs and 22 non-polio viruses (Table 1).
157
The seasonal distribution of PVs and NPEVs during the study is presented in Figure 1. After
158
re-isolation of a single PV serotype by the NT assay, we conducted sequence analysis of the PV
159
capsid VP1 gene. The serotype frequency of PV-positive isolates was 23 type 1 (PV1), 38 type 2
160
(PV2), and 22 type 3 (PV3) (Table 1). Sequence analysis of the full-length VP1 region showed that all
161
isolates were Sabin-like, with 1% nucleotide divergence from Sabin 1 and Sabin 3 and 0.6%
162
divergence from Sabin 2 (32). As shown in Table 1, the frequency of PV-positive isolates obtained at
163
the T plant was >1.5-times higher than that of isolates obtained at the Y plant. The distribution of each
164
serotype showed a similar trend, with a greater frequency at the T plant than at the Y plant. These
165
distributions mirrored the municipal vaccination (OPV) period in each area for approximately 2–3
166
months (Fig. 1). The frequency of isolated PVs was highest in 2010, and tended to gradually decrease
167
in the successive months of the sampling period. The last isolation of PV was in May 2012 at the T
168
plant and November 2011 at the Y plant. Since September 2012, routine IPV immunization is
169
conducted nationwide. Thus, before the immunization transition, PV originating from OPV had
170
disappeared from sewage water at both plants. On the other hand, NPEVs have been isolated over
171
research period (data not shown).
172
8
173
Nucleotides substitutions on VP1 region
174
The use of primers specific to the VP1 region of each PV serotype revealed mutations in the viral
175
capsid sequence of the PV isolates, relative to the Sabin vaccine strains. Of the 83 PV isolates
176
obtained during this study, 69 (83%) showed at least one mutation; two PV3 variants had six
177
nucleotide mutations in VP1 (Fig. 2).
178
With regard to the mutations detected in the VP1 region, we closely examined the
179
nonsynonymous mutations affecting the codons of amino acid residues. Many of these mutations are
180
“attenuation markers” responsible for the attenuation phenotype of OPV strains (4). Several groups
181
reported the following markers in the VP1 region of the Sabin vaccine strains: T106 and F134 in Sabin 1
182
(S1) (33), I143 in Sabin 2 (S2) (34), and T6 in Sabin 3 (S3) (35, 36). In the VP1 sequence of the 23 S1
183
isolates, we found 12 isolates with an amino acid substitution at T106 (52%) (Fig. 3). All these
184
mutations exhibited nonsynonymous mutations T → A/S at residue 106 from the reference S1 strain.
185
In contrast, no modified codon was found in the F134-coding region of the S1 isolates. Among the 38
186
S2 isolates, we found 20 isolates with the mutation at the I143 residue (53 %). These mutations also
187
included three types of nonsynonymous substitutions (I143 → T/V/N) affected by the first and second
188
nucleotide modifications of this codon. On the other hand, OPV Sabin 3 strain has been known to
189
contain mutations at the second codon of the 6th amino acid position of VP1 even in the
190
manufacturing. In fact, all PV3 isolates had isoleucine at the 6th position unlike original Sabin 3
191
having threonine. In this study, we described the amino acid change as mutation. In summary,
192
molecular analysis of the isolated PV serotypes revealed that several isolates contained mutations
193
affecting amino acid residues of known “attenuation markers” of VP1.
9
194
DISCUSSION
195
Environmental surveillance of PVs is critical to maintain polio-free areas and to work toward the
196
global eradication of polio. We present the results of a 34-month environmental virus surveillance
197
study, conducted at two independent sewage disposal plants located in urban and rural areas of
198
northern Kyushu district, Japan. In total, 83 Sabin-like PVs were isolated from wastewater samples
199
collected monthly from April 2010 to January 2013. The surrounding areas of each plant had unique
200
features, including differences in sewage line coverage and infant age distribution, despite the similar
201
population size (Table 1). We sought to determine the frequency of PV isolation from wastewater
202
around the OPV to IPV transition period in those communities.
203
In Japan, the usage rate of disposable diapers is quite high (approximately 80%–90%). If
204
disposable diapers containing stool from OPV-immunized children are properly treated and discarded,
205
the frequency of PV detection in sewage water would presumably be quite low. However, PV was
206
detected in wastewater sampled from two areas (T and Y disposal plants) with scheduled OPV
207
immunizations and different infant age distributions. This finding suggests that either stool from
208
OPV-immunized children flowed into the sewage line and contaminated the water or intrafamilial
209
OPV transmission led to PV excretion into the sewage water. In fact, several reports have indicated
210
intrafamilial and interfamilial spread of PV, including the vaccine strain and NPEV (37-39).
211
Because of the high usage rate of disposable diapers, PVs from OPV-immunized children may be
212
transmitted by familial contacts, such as siblings and parents, or contacts at school facilities, such as
213
kindergarteners (40). However, we would also anticipate NPEV to be widely transmitted among
214
family members. In Toyama, Japan, a correlation was established between the high frequency of
215
echovirus type 13 (E13) isolation from environmental water in the summer and seroconversion
216
against E13 between preoutbreaks and postoutbreaks, regardless of age (41). These studies suggest
10
217
that we should expect differences in the frequency of NPEV between the two plants. On the other
218
hand, we showed that the frequency of PV was clearly related to the OPV vaccination period.
219
Previous studies have reported a rapid decline in PV isolation around the OPV to IPV transition
220
period, with a disappearance of PV vaccine strains from wastewater within 2 to 3 months after the
221
cessation of OPV administration (42-45). In contrast, our study showed the disappearance of
222
Sabin-like PVs from the environment before OPV immunization had ceased (Fig. 1). PVs have not
223
been detected from wastewater at the T and Y plant since June 2012 and December 2011, respectively.
224
After the announcement that IPV would replace OPV for routine immunization in Japan (May 2011),
225
the nationwide coverage by routine OPV immunization declined until IPV was introduced in
226
September 2012, mostly because of public concerns about VAPP (12, 13). The rate of VAPP is
227
assuming 1 per two to five million inoculations in Japan. However, the rate might not be low for the
228
parents have children should be received polio vaccine in the period. And from now on, it will be very
229
important to monitor the decline and disappearance of VAPP cases after OPV cessation, as
230
demonstrated in USA (46).
231
The analysis of the viral capsid VP1 sequence showed that all PV isolates were Sabin-like
232
vaccine strains. Many isolates contained mutations in the VP1 sequence. However, major deviations
233
from the attenuated Sabin strains were relatively rare, and there was no significant difference in
234
mutations between the virus serotypes. Importantly, these data show that no VDPV has emerged and
235
circulated in the sampling sites over the course of this study.
236
The PV1 isolates also presented several mutations affecting codons and amino acid residues,
237
known as attenuation markers, in the VP1 region of all PV serotypes. In the Sabin 1-like isolates, the
238
T → A amino acid mutation at position 106 was the most frequent. This mutation introduces a residue
239
found in the Mahoney strain, a neurovirulent parent of the Sabin 1 strain. With regard to the Sabin
11
240
2-like isolates, the I143 position was mutated (I → T/V/N) in 53% of the isolates. All Sabin 3-like
241
strains isolated in this study had isoleucine at the 6th position of VP1 amino acid unlike the original
242
Sabin 3 strain having threonine. It is well known that Sabin 3 of OPV originally has mixed
243
nucleotides at second codon of the relevant position even in manufacturing (47). But it is difficult for
244
us to know at what stage the mutations occurred. And our study have not intended to show the
245
virulence and replication ability of isolates but just the numbers of VP1 mutation for denying the
246
emergence of VDPV in the research field. This is one of the limitation points of this study. Besides
247
this, the strains have isolated from sewage water not from patients and other healthy persons. That’s
248
why we can only speculate when the mutations occurred and the effect of VP1 mutations to virulence
249
of the isolates. More information may be obtained when we sequence genome of the isolates and
250
compare it with known marker of other regions. However, our results indicate that the continuous
251
surveillance of nucleotide substitutions is necessary to monitor the emergence of VDPVs and other
252
mutation strains.
253
In conclusion, we offer an environmental surveillance strategy that can detect the excretion of
254
PVs from OPV-immunized populations with high sensitivity. Needless to say, the transition from OPV
255
to IPV is an essential step toward the global eradication of PV. However, the transition from OPV to
256
IPV should be carefully orchestrated before OPV cessation to maintain immunity coverage by OPV.
257
Even if the IPV transition is successful, the risk of PV infection for a susceptible population must be
258
closely monitored with a high-quality surveillance system because PV can be silently transmitted and
259
its viral genome can mutate during replication in the human gut, partly because of insufficient
260
mucosal immunity (48, 49). Under these circumstances, the environmental surveillance of PV plays a
261
key role not only to monitor the importation of wild-type PV from endemic areas but also to prepare
262
against emerging VDPVs before the global cessation of OPV at the final stage of polio eradication.
12
263 264
Acknowledgement
265
This study was supported by a grant for Research on Emerging and Re-emerging Infectious
266
Diseases from the Ministry of Health, Labour and Welfare of Japan. We appreciate the technical
267
suggestions of Drs Takenori Takizawa and Masae Iwai-Itamochi from the Toyama Institute of Health
268
and the critical comments of Drs Hiroyuki Shimizu and Chikako Kataoka from the National Institute
269
of Infectious Diseases, Japan.
13
270
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458
Figure Legends
459 460
Figure 1. Frequency of poliovirus (PV) and non-polio enterovirus (NPEV) isolation at two
461
independent sewage plants
462
Plants T and Y were compared for the frequency and distribution of NPEV and poliovirus type 1, type
463
2, and type 3 isolated over the 34-month collection period. The double-headed arrows above each
464
graph indicate scheduled vaccination periods in each area. The dotted vertical line indicates the month
465
and year when IPV was introduced in Japan. The first Y axis (left) indicates the frequency of PV
466
isolation each month, and the second Y axis (right) indicates the frequency of NPEV isolation each
467
month. Sample collection was conducted from April 2010 to January 2013 (X axis).
468 469
Figure 2. Distribution of nucleotide substitutions in the VP1 regions of the isolated strains of
470
polioviruses (PV)
471
The nucleotide divergence in VP1 between isolates and reference vaccine strains (Sabin 1: AY082688,
472
Sabin 2: AY082679, and Sabin 3: AY082683) is shown on the X axis. The number of isolates with
473
each mutation is shown on the Y axis.
474 475
Figure 3. Nucleotides substitutions on VP1 region
476
Above each nucleotide, the column indicates the percentage of all PVs of an isolated serotype during
477
the collection period that featured a mutation at this position. Below each codon, the circle shows the
478
amino acid substitution resulting from the mutation. The letters below the nucleotides and capital in
479
the circles represent conventional abbreviations for amino acids. *The OPV Sabin 3 originally
480
contains mutation at the second codon of VP1 6th amino acid known as “mix base position”.
20
Table 1
Table 1 Comparison of two sewage plants appearance and isolated serotype of poliovirus
Isolated serotyope (%)a
Environment
Watershed population
0-2 years old populaton
Sanitation Coverage
CPE-positive
L20B CPEpositive
PV isolates (NT)
PV1
PV2
PV3
T plant
urban
190,000
2,300
≥ 90%
248
67
52
16 (31)
25 (48)
11 (21)
Y plant
rural
180,000
1,400
≤ 10%
198
36
31
7 (23)
13 (42)
11 (35)
Total
-
-
-
-
446
103
83
23 (28)
38 (46)
22 (27)
a) % are calculated from the numbers of all isolated PVs of the area
