Research On The Regional Circulation And Molecular Epidemiologyof Human Enteroviruses Based On Environmental Surveillance

[Background]Enteroviruses (EV) are common viruses living in human intestinal and are associated with a variety of clinical symptoms. They usually cause asymptomatic infection and wild diseases, but also can lead to serious or deadly diseases. Recent studies showed that EV infection was also associated with certain chronic diseases, such as type I diabetes and thyroid inflammation, etc.Enteroviruses spread widely throughout the world without geographical restrictions. With the rapid growth in international exchanges, the magnitude of EV infection is beyond our expection and the changing environment is likely to accelerate the variation of the viruses. Diseases caused by enteroviruses usually are sporadic or regional outbreaks. In non-epidemic period, there are several co-circulating dominant serotypes in specific area while a large number of strains of same type will be separated as outbreaks occur. EV infection is a serious threat to the individuals especially children’s health and life safety and become one of major public health problem for a long time. To strengthen EV molecular epidemiological surveillance has important theoretical and practical significance for prevention and control of EV-related diseases.EV infection is chiefly latent infection and once been infected, patients and individuals with latent infection will continually excrete large amounts of viruses to environment for several weeks. Enteroviruses in the environment can be concentrated, separated and identified by different experimental methods as supplement of EV epidemiological studies, known as environment surveillance. Sewage is a rich source of enteroviruses circulating in a specific area. Environmental surveillance could provide the information of EV circulation in a particular region, including symptomatic and latent infection and is possible to detect EV in the sewage before crowd clinical symptoms appear. EV is widespread in environmental water and can be separated in a variety of environmental water. But viral titer is relatively low, and EV is easily attached to the suspended particles, so it is harder than the detection of clinical specimens. Therefore, environment samples must be concentrated and enriched before testing and it’s often the key point to the success of separation. Anion membrane absorption-elution method is widely applied in current research and plays an important role in EV environment surveillance as well as the monitoring of related disorders pathogens.Enteroviruses are one of research focuses in related fields and extensive epidemiological and virological studies are conducted in recent years. Currently, a number of countries carry out environmental surveillance to understand the local polio virus and non-polio enteroviruses circulation status and conduct related studies, but domestic research in this field is little.[Objectives]1. To continue to optimize anion membrane absorption-elution method of environment sewage samples and to improve the efficiency of detection on the basis of the previous studies. To discuss the virus adsorption and influencing factor of suspended particles in environment sewage.2. To clear explicitly enterovirus serotypes and distribution in Shandong province from 2012 to 2014 and provide support to related epidemiological studies.3. To add and perfect the VP1 gene database of serotypes isolated from environment sewage samples in Shandong province, to research the genetic characterization and long-time cycle dynamic changes of EV in Shandong province and to provide valuable support for enterovirus epidemiological studies.[Methods]1. Environment surveUlance: We chose Jinan Everbright sewage treatment plant and Linyi shouchuang treatment plant as sampling sites,and then Yantai Xi,an River treatment plant was chosen in September 2013. Sewage samples were collected at regular intervals at the entrance of sewage and concentrated with anion membrane absorption-elution method. RD and Hep-2 cells were used in virus isolation.2. Case Surveillance: APP and AMES survdllance system chosen in the study.3. Amplication of gene and sequence determination: Viral RNA was extracted and VP 1 coding region was amplified by RT-PCR and sequenced. Isolates were typed by molecular typing method.4. Biological information analysis of EV: Homologous analysis on VPl sequences of environment and case surveillance and others submitted in GenBank were conducted by Bioedit 7.0.9.0. Phylogenetic analysis constructed by the neighbor-joining method based on the alignment of the VPl gene sequences of dominant serotypes in environment and case surveillance and the corresponding strains from GenBank were performed by MEGA 5 .0.[Results]1. Optimization of anion membrane absorption-elution method Recovery rate of EV by eluent of nent pH values and different elution times of ultrasonic oscillations were compared. The conditions of elution were optimized: pH value of eluent was 9.0 and the elution time of ultrasonic oscillations was 3 minutes.2. EV adsorption of suspended particle in environment sewage While the sewage is acidic, it is beneficial to virus adsorption to suspended particle; the content of EV has no effect on the adsorption effect of suspended particle in the actual cases.With the increase of suspended particle in sewage,adsorption rate is on the rise while the virus elution rate has obviously downward trend,the overall recovery efficiency in certain concentration range remain stable. The suspended particle in environment sewage samples has strong adsorption with E-3 and weak adsorption with E-6.3. Sampling and isolation of EV in environment and case surveillance We isolated EV 2010 strains in environment surveillance samples from 2012 to 2014, including NPEV 1468 strains and major dominant serotypes are E-7, CV-B5, E-6, E-3, E-11, CV-B3; PV 542 strains involved of a VDPV type 2 strain E12-221 and other 12 pre-VDPV. Different cities had different serotype spectrum in different months and different serotypes had different time circulation mode. We separated NPEV 119 strains in AFP case surveillance, including 27 types; PV 35 strains, no VDPV isolates and PV wild strains. In AMES surveillance system in 2014, we collected 798 samples and isolated EV 152 strains, the positive rate is 19.1%, CV-B5, E-6 and E-30 were dominant serotypes.4. Phylogenetic analysis of dominant strains in environment and case surveillance E-7 isolated most of all serotypes and had low homology between the isolates which suggested that more than one transmission chains of E-7 was co-circulating. E-3 had low speed of nucleotide and amino acid mutation and kept stable with obvious time and regional clustering. CV-B5 isolates had high level of homology and two transmission chains exsit. It spreaded quickly and was widely prevalent in Shandong province. Conversely, CV-B3 had low level of homology and distinct time clustering. There were great nucleotide differences between E-6 isolates and at least two transmission chains exsit in the province. E-11 included Gergory-like strains and Silva-like strains and divided into two evolutionary branchs with great differences between them.[Conclusions]1. According to recovery experiments, we determine the optimal elution conditions of anion membrane absorption-elution method by adjusting pH value of eluent and the elution time of ultrasonic oscillations.2. Suspended particles in environment sewage have strong adsorption of EV, and influenced by many factors, and EV separation in suspended particles has little difference with it in the supernatant. In the future environment surveillance, we can use both of them in the research.3. During this research, we isolated many serotypes of enterovirus group B in the environment surveillance and case surveillance, while serotypes of enterovirus of group A and group C were very little and no group D was found. The dominant serotypes of NPEV isolates were E-7, CV-B5, E-6, E-3, E-11 and CV-B3. Different cities have different serotype spectrum in different months and different serotypes have different time circulation mode.4. We didn’t find PV wild strains in PV environment sewage samples, but found a VDPV tyoe 2 strain E12-221 for the first time in domestic environment surveillance.5. By homology comparison and phylogenetics analysis, we found that the EV dominant serotypes in Shandong Province evolved fast and the mutation of them was active. There are several transmission chains of each serotype. The dominant serotypes in environment surveillance and case surveillance has certain genetic evolutionary relationships.6. Through the analysis of environment sewage samples, the high sensitivity of environment monitoring of EV was proved. It can obtain the EV prevalence and time circulation pattern in the external environment and also can be used as a complementary means to evaluate PV prevalence besides AFP surveillance system. Environment surveillance can provide scientific basis for prevention and control related diseases.