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Advance Publication by J-STAGE Advance Publication by J-STAGE Japanese Journal of Infectious Diseases 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 Received: July 27, 2017. Accepted: June 28, 2018 Published online: July 31, 2018 DOI:10.7883/yoken.JJID.2017.338 Advance Publication articles have been accepted by JJID but have not been copyedited or formatted for publication. 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, Zhaodan Sun, Expanded Program on Immunization, Heilongjiang Provincial Center for Disease Control and Prevention, Harbin Corresponding author: Mailing address: Institute of Planned Immunity, Heilongjiang Center for Disease Control and Prevention, 40 Youfang Street, Xiangfang District, Harbin, Heilongjiang Province, China, 150030. Tel&Fax: +86-451-55153604, E-mail: [email protected] Key words: Poliovirus, Environmental Surveillance, VP1, Genetic Mutation Running Title: Poliovirus detection and genetic characteristic analysis Accepted Manuscript Summary: By monitoring sewage in Heilongjiang Province from 2013 to 2016, in this research we analyzed the epidemiological tendency and genetic mutation found in the poliovirus in the environment in order to setup a warning system for vaccine-derived poliovirus (VDPV) and spread of wild poliovirus. In this study, we collected 139 sewage samples from eight regions in Heilongjiang Province. Poliovirus was identified from 72 samples and the positive rate was 51%. 263 strains of poliovirus were isolated, including 22 type1, 104 type2 and 137 type3 polioviruses. As results of intratypic differentiation (ITD), using real time PCR and nucleotide sequencing, three type 1 pre-VDPV, one type 2 VDPV and two type 3 pre-VDPV strains were isolated, respectively. Interestingly, one type 1 strain with 5 nucleotide deletion and one type 3 recombinant on VP1 were isolated. By monitoring the poliovirus in the environment continually, we could recognize the VDPV or wild poliovirus with the high neurovirulence from large-scale circulation and set up warning system to avoid morbidity and transmission of the virus. Introduction Poliovirus (PV)es mainly spread by fecal-oral transmission, and the PV mostly causes inapparent infection after infecting human hosts. However, in a few cases out of a thousand infected, PV can cause numbness, even permanent disability. Thanks to the application of oral poliomyelitis attenuated live vaccine (OPV) around the world, effort of poliovirus’ eradication has made significant progress, but in some neighboring nations (Pakistan and Afghanistan) of China, there are still transmissions of wild poliovirus (WPV) which have not been blocked at all. Hence, China still has the risk of cross-border transmission of WPV (1,2). Poliovirus vaccine strain undergoes gene mutation and genetic recombination during the replication process in human body. Revertant mutations happening in some attenuation positions may lead to a rise ofAccepted neurovirulence, which are known Manuscript to have caused several outbreaks of paralytic poliomyelitis in different areas of the world (3,4). Therefore, the existence of vaccine-derived poliovirus (VDPV) causes more and more attentions in many countries. PV has a strong ability to survive in environment, and a large number of viruses excreted into the environment will survive for a certain period and keep infectious (5). Continuous monitoring of external environment is helpful to grasp the distribution of the poliovirus in the environment and also to detect WPV and circulating VDPV (cVDPV) in time, and it can provide solid basis for prevention and control of poliovirus outbreak. Materials and Methods 1. Monitoring area, sampling frequency and the sample quantity between 2013-2016 As the capital city of Heilongjiang Province, Harbin city has vast floating population so there is the need of monitoring PV status in environment. We set major urban area and Shuangcheng County to be two sampling sites. In urban area of Harbin City, samples were collected once a month and total of 48 samples were collected. In Shuangcheng County, 40 sewage samples were collected. Along the border area and the area attacked by flood in 2013, 6 sampling sites were chosen, where high risk of WPV spread was. And samples were collected in summers because the sewage water in summer was more suitable for the polioviruses to survive. In Fuyuan county of Jamusi City, Tongjiang County of Jiamisi City, and Mudanjiang City, there were 9 samples collected in each area. In Aihui District of Heihe City, 12 samples were collected. In Zhaoyuan County of Daqing City and Suifenhe county of Mudanjiang City, there were 6 samples collected respectively(see Table1.). The distribution of sampling sites was illustrated in Figure 1. One Liter of sewage water was collected from the entry of local sewage treatment plants at each sampling sites to proceed the environmental monitoring work. 2. Sample transportation and treatment Within 48 hours, all the sewage samples were placed in refrigerated transportation and sent to the polio laboratory of Heilongjiang provincial center for disease control and prevention (Heilongjiang CDC), and the sewage samples were concentrated and enriched by using the filter adsorption and elution method. 500mL of each samples were centrifugedAccepted under 4℃ with 3000 rpm Manuscript for 30 minutes. 2.5 M MgCl2 solution was added into the supernatant to make a final concentration of 0.05 M and 0.5 N HCl was used to adjust total pH value to 3.5.A cellulose nitrate membrane filter (0.45μm pore size; model No.31023150; Toyo Roshi, Tokyo, Japan) was used as a virus adsorbent through multi-position filtration system (BioVac321B; Chemvak, Germany). The membranes were cut into pieces after collection. The viruses adsorbed on the membrane fragments were eluted into 10ml of 3% beef extract solution with the pH value of 9 by using vortex mixer. After the collecting of eluent, another 10ml of 3% beef extract was added into the rest of the membrane fragments for the second elution process. The first eluent and the second eluent were centrifuged respectively under 3000rpm for 30 minutes. The supernatant was collected after filtration with 0.45μm filter for the use of virus isolation experiments.(6) 3. Isolation and identification of the poliovirus The sewage concentrates were inoculated respectively in Human Rhabdomyosarcoma (RD) cells, Mouse Cell line Expressing the Gene for Human Cellular Receptor for Poliovirus (L20B) and Human Laryngeal Carcinoma (HEp-2) with full of monolayer cell to detect and isolate the poliovirus. The first eluent was inoculated in 5 tubes of each cell line, and the second eluent were inoculated in 3 tubes respectively. For each tube, 200 μL eluent was inoculated. The Cultures in which Characteristic enterovirus cytopathic effect (CPE) was observed in RD or HEp-2 cells were re-inoculated onto L20B cells. Positive L20B isolates were collected and the poliovirus type-specific polyclonal antisera (Produced by RVIM, provided by WHO and polio laboratory of China CDC) was used to make identification (7). All PV isolates were confirmed by intratypic differentiation (ITD) (PV isolates 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). 4. Sequencing of VP1 Region. 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 national criteria for originated strain of AcceptedSabin vaccine was used to assess Manuscriptthe risk of polio case, according diagnosis for poliomyelitis in China: Sabin-Like, pre-VDPV and VDPV. Comparing the sequencing result with Sabin strain: VP1 coding region nucleotide sequence variations in VDPV of type1 and 3 ≥10，and ＜135（mutation rate＞1%，and＜15%)，in VP1 coding region nucleotide sequence variations of VDPV of type2 ≥6，and ＜135 （mutation rate ＞0.6%，and＜15%). For VP1 coding region nucleotide sequence variation of type1 and type3 are between 6 and 9, they could be judged to be high mutant strain (pre-VDPV) (8). PV strain which was unqualified for mutation standards of VDPV and pre-VDPV were classified as Sabin-Like (SL). 5. Sequence Analysis Sequencher 5.2.4 software was used to analyze the sequencing results. In the comparative analysis, as reference, Sabin strains of type 1,2 and 3 had completed sequences on Genbank respectively：AY184219.1, AY184220.1 and AY184221. And VP1 sequence codes of type 1,2 and 3 Sabin strains on Genbank were respectively: AY082688.1、AY082679.1、AY082683.1. Results 1. PV isolation of Sewage in External Environment Between 2013 and 2016, there were total 139 sewage samples collected from monitoring sites around Heilongjiang province, and there were 72 samples detected PV with positive rate of 51.8%. Within all, there were 82 samples detected containing Non-Polio Enterovirus (NPEV), with positive rate of 59.0%. Among the sewage samples, 263strains of PV and 312 isolates of NPEV were isolated, including 22 strains of type 1 PV (PV1), 104 strains of type 2 PV (PV2) and 137 strains of type 3 PV (PV3). The monitoring sites were shown in Table 1. 2. Mutations in PV detected from sewage samples According to the update definitions of VDPV and pre-VDPV(8), during 2013-2016, there were totally 1 strain of type 2 VDPV, 3 strains of type 1 pre-VDPV, 2 strains of type 3 pre-VDPV, and 1 strain of PV1 with gene deletion and 1 strain of type 3+2 with gene recombination. Other strains of PV were identified to be SL. Sampling sites which VDPV and pre-VDPV were isolated from were shown in Figure 1 and mutation details wereAccepted shown in Table2.
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