Jpn. J. Infect. Dis., 71, 442–447, 2018 Original Article Poliovirus Detection and Genetic Characteristic from Sewage in Heilongjiang Province from 2013 to 2016 Yujie Ma*, Xiaoyu Wang, Xue Zhou, Po Lv, Han Wang, Jing Song, Changjiang Song, and Zhaodan Sun Institute of Planned Immunity, Heilongjiang Center for Disease Control and Prevention, 40 Youfang Street, Xiangfang District, Harbin, Heilongjiang Province 150030, China SUMMARY: By monitoring the sewage system in Heilongjiang province from 2013 to 2016, this study aimed to analyze the epidemiological tendency and genetic mutation of poliovirus (PV) found in the ­environment in order to setup a warning system for vaccine-derived poliovirus (VDPV) and the spread of wild poliovirus. In this study, we collected 139 sewage samples from 8 regions in Heilongjiang province. Poliovirus was identified from 72 samples, and the positivity rate was 51%. A total of 263 PV strains were isolated, including 22 strains of type 1 PV, 104 strains of type 2 PV, and 137 strains of type 3 PV. As a result of intratypic differentiation, using real-time PCR and nucleotide sequencing, 3 type 1 pre-VDPV, one type 2 VDPV, and 2 type 3 pre-VDPV strains were isolated. Interestingly, one type 1 strain with 5 nucleotide deletions and one type 3 recombinant on VP1 were isolated. By continuously monitoring the poliovirus in the environment, we aimed to recognize the VDPV or wild poliovirus with high neuroviru- lence from large-scale circulation and set up a warning system to avoid morbidity and virus transmission. tion of WPV and circulating VDPV (cVDPV). In addi- INTRODUCTION tion, it can provide a solid basis for prevention and con- Polioviruses (PVs) mainly spread by fecal-oral trans- trol of poliovirus outbreak. mission, and PVs usually cause inapparent infection after infecting a human host. However, in some cases, PV can MATERIALS AND METHODS cause numbness or even permanent disability. Thanks to the widespread use of oral poliomyelitis attenuated live Monitoring area, sampling frequency, and sample vaccine (OPV), the global effort to eradicate poliovirus quantity between 2013 and 2016: As the capital city of has made a significant progress. However, in some Heilongjiang Province, Harbin city has a vast floating neighboring countries of China (such as in Pakistan and population; hence, the status of PV found in the environ- Afghanistan), there are still transmissions of wild polio- ment should be monitored. We set major urban area and virus (WPV) which have not been blocked at all. Hence, Shuangcheng County as the 2 sampling sites. In the China remains at risk of cross-border transmission of ­urban area of Harbin City, samples were collected once a WPV (1,2). A poliovirus vaccine strain undergoes gene month and a total of 48 samples were collected. In mutation and genetic recombination during the replica- ­Shuangcheng County, 40 sewage samples were col- tion process in the human body. Revertant mutations lected. Along the border area and the area that was af- ­occurring in some attenuated positions may increase fected by flood in 2013, 6 sampling sites, with higher neurovirulence, which are known to have caused several risk of WPV spread, were chosen. The samples were outbreaks of paralytic poliomyelitis in the different parts ­collected in summer because the sewage water during of the world (3,4). Therefore, the emergence of vaccine- this season was more suitable for poliovirus survival. In derived poliovirus (VDPV) is gaining more attention in Fuyuan county of Jiamusi city, Tongjiang county of many countries. PV has a strong ability to survive in the ­Jiamusi city, and Mudanjiang city, 9 samples were col- environment, and a large number of viruses excreted into lected in each area. In Aihui District of Heihe city, 12 the environment will survive for a certain period and re- samples were collected. In Zhaoyuan county of Daqing main infectious (5). Continuous monitoring of the exter- city and Suifenhe county of Mudanjiang city, 6 samples nal environment is helpful to understand the distribution were collected (Table 1). The distribution of sampling of poliovirus in the environment and for the early detec- sites is illustrated in Fig. 1. One liter of sewage water was collected from the entry point of local sewage treat- ment plants at each sampling site in continuation with the environmental monitoring work. Received July 27, 2017. Accepted June 28, 2018. Sample transportation and treatment: Within 48 h, J-STAGE Advance Publication July 31, 2018. all the sewage samples were placed in refrigerated trans- DOI: 10.7883/yoken.JJID.2017.338 portation and sent to the polio laboratory of Heilongjiang *Corresponding author: Mailing address: Institute of Provincial Center for Disease Control and Prevention Planned Immunity, Heilongjiang Center for Disease Con- (Heilongjiang CDC), and the sewage samples were con- trol and Prevention, 40 Youfang Street, Xiangfang District, centrated and enriched using the filter adsorption and Harbin, Heilongjiang Province 150030, China. Tel: +86- elution method. Approximately 500 mL of each sample 451-55153604, E-mail: [email protected] was centrifuged at 3,000 rpm for 30 min at a temperature 442 Poliovirus Detection and Genetic Characteristic Analysis Table 1. Environment surveillance situation in Heilongjiang province between 2013 and 2016 Sample Sample No. of Sampling Sample PV+ Type 1 Type 2 Type 3 Total Sampling period no. of no. of NPEV sites no. rate% strains strains strains strains PV+ NPEV+ isolate Haerbin Jan. 2013–Dec. 2016 48 32 66.7 18 53 58 129 38 135 Shuangcheng Sep. 2013–Dec. 2016 40 15 37.5 0 22 13 35 13 45 Heihe Jul., Aug., Sep. of 2013–2016 12 7 58.3 1 6 13 20 8 15 Fuyuan Jul., Aug., Sep. of 2013, 2014 and 2016 9 6 66.7 0 17 31 48 2 13 Tongjiang Jul., Aug., Sep. of 2013, 2014 and 2016 9 4 44.4 1 0 8 9 6 35 Apr., May, Jun. of 2013; Mudanjiang 9 3 33.3 0 5 6 11 5 19 Jul., Aug., Sep. of 2013–2014 Suifenhe Jul., Aug., Sep. of 2013 and 2016 6 3 50.0 0 0 5 5 6 42 Zhaoyuan Jul., Aug., Sep. of 2014 and 2016 6 2 33.3 2 1 3 6 4 8 Total — 139 72 51.8 22 104 137 263 82 312 rus cytopathic effects in RD or HEp-2 cells were re- inoculated onto L20B cells. Positive L20B isolates were collected, and the poliovirus type-specific polyclonal an- tiserum (produced by RVIM, provided by the WHO and China CDC polio laboratory) was used for identification (7). All PV isolates were confirmed by intratypic differ- entiation (ITD). PV isolates collected before December 2014 were shipped to China CDC for ITD confirmation process and after January 2015, ITD for PV isolates was conducted in Heilongjiang CDC. Sequencing of VP1 region: The primer set of UG1/ UC11 was used for amplification of VP1 region with the sequence of UG1:5′-TTTGTGTCAGCGTGTAATGA- 3′, and UC11:5′-AAGAGGTCTCTATTCCACAT-3′. The sequencing work was conducted by Sangon Biotech (Shanghai) Co., Ltd. Three levels of the national criteria for originated strain of Sabin vaccine were used to assess Fig. 1. Distribution of sampling sites and poliovirus strains iso- lated in Heilongjiang Province. Each dot indicated one VDPV the risk for polio. Three levels of diagnosis for poliomy- or pre-VDPV strain. elitis in China are Sabin like (SL), pre-VDPV, and VDPV. The sequencing results of Sabin strains were compared as follows: in the VP1 coding region, the of 4°C. About 2.5 M of MgCl2 solution was added into ­nucleotide sequence variations of VDPV types 1 and 3 the supernatant to make a final concentration of 0.05 M, were ≥ 10 and < 135 (mutation rate: > 1% and < 15%), and 0.5 N HCl was used to adjust the total pH value to respectively, while those of VDPV type 2 were ≥ 6 and 3.5. A cellulose nitrate membrane filter (0.45-μm pore < 135 (mutation rate: > 0.6% and < 15%). In the VP1 size; model no. 31023150; Toyo Roshi, Tokyo, Japan) coding region, if the nucleotide sequence variations of was used as a virus adsorbent utilizing a multi-position types 1 and 3 were between 6 and 9, they were judged as filtration system (BioVac321B; Chemvak, Germany). high-mutant strains (pre-VDPV) (8). PV strains whose The membranes were cut into pieces after collection. mutation standards did not qualify for VDPV and pre- The viruses adsorbed on the membrane fragments were VDPV were classified as Sabin like (SL). eluted into 10 mL of 3% beef extract solution with a pH Sequence analysis: The Sequencher 5.2.4 software value of 9 using a vortex mixer. After the collection of was used to analyze the sequencing results. In the com- eluents, another 10 mL of 3% beef extract was added parative analysis, Sabin 1, 2, and 3 strains had completed into the rest of the membrane fragments for the second sequences on Genbank respectively: AY184219.1, elution process. The first eluent and the second eluent AY184220.1 and AY184221. The VP1 sequence codes were centrifuged at 3,000 rpm for 30 min. The superna- of Sabin 1, 2, and 3 strains on GenBank were tant was collected after filtration using a 0.45-μm filter AY082688.1, AY082679.1, and AY082683.1, respec- for virus isolation experiments (6). tively. Isolation and identification of the poliovirus: The sewage concentrates were inoculated in human rhabdo- RESULTS myosarcoma (RD) cells, mouse cell lines expressing the human cellular receptor gene for poliovirus (L20B), and Isolation of PVs from sewage samples collected human laryngeal carcinoma (HEp-2) filled with mono- from the external environment: Between 2013 and layer cells to detect and isolate the poliovirus.