Concentration And Isolation Of Human Enteroviruses From Domestic Sewage And Application Research In Environmental Surveillance

【Background】Human enteroviruses (HEVs) belong to family Picornaviridae. They mostly cause self-limited infections, but may also cause a variety of clinical dieases such as acute flaccid paraiysis (AFP), aseptic meningitis (AM), myocarditis, acute hemorrhagic conjunctivitis (AHC), hand, foot and mouth disease (HFMD), neonatal systemic infection and even death.HEVs were traditionally divided into poliovirus (PV), coxsakieviruse A (CVA), coxsakieviruse B (CVB) and Echovirus (Echo). In November,2011, International Committeeon Taxonomy of Viruses (ICTV) published the newest report of virus classification, which included human rhinovirus (HRV) into the genus Enterovirus and HEVs are divided into7groups, HEV-A-HEV-D and HRV-A-HRV-C.HEVs have a unique way to excrete toxins to the environment. Once been infected, individuals with clinical symptoms or not will continually excrete feces with virus for several weeks. The virus can survive and keep infectious for different time according to the environment conditions. During this period, sewage samples can be collected for virus concentration, isolation and identification by distinct experimental methods.Usually, the titers of HEVs are extremely low in sewage, which can not meet the needs of direct detection. Therefore, virus concentration is an important issue. So far, methods for virus concentration from sewage samples are mainly adsorption-elution method, membrane filtration and chemical flocculation, et al. The first two methods are widely used in the concentration and detection of virus. This study will combine these two methods, using anion membrane adsorption under high magnesium ions and low pH condition, and then adding eluent, followed by eluting the virus with ultrasonic vibration.During the global polio eradication program, AFP surveillance system can provide a distribution baseline of PV and other non-polio enterovirus (NPEV) in a given region. However, the data can not reflect the epidemic status of HEVs due to the low AFP incidence, as well as asymptomatic infections. Environmental surveillance is the most sensitive way for detecting wild poliovirus and vaccine strains. What is more, it is also an effective way to trace prevalent and minor enterovirus circulating in the human population. Therefore, since our contry had achieved the target of polio eradication, optimizing the methods of HEVs concentration from sewage and using these methods in the application research of environmental surveillance of PV and other NPEV, could be significant in confirming polio eradication, tracing NPEVs that cause silence infection as well as predicting the incidence of disease.【Objective]1. To optimize the methods of HEVs concentration from sewage;2. To explore the distribution of HEVs in the environment and genetic variation ofPVs and NPEVs;3. By comparing the genetic characterization between clinical NPEVs and environmental isolates, to predict the outbreak of related diseases and to evaluate the significance of environmental surveillance in tracing VDPV, WPV.[Methods]1. Jinan everbright sewage treatment plant and Linyi shouchuang treatment plant were chosen as sampling sites. Sewage samples were collected at regular intervals, and concentrated with anion membrane-ultrasonic vibration; 2. RD, Hep-2and L20B cells were used in virus isolation. Neutralization test was used to identify the serotype of enteroviruses.3. Viral RNA was extracted. VP1coding region was amplified by RT-PCR and sequenced. Isolates were typed by molecular typing method.4. Homologous analysis on VP1sequences from environmental surveillance, AFP sueveillance, AM patients, and others submitted in GenBank were conducted using BioEdit7.1.3;5. Phylogenetic analysis constructed by the Neighbor-Joining method based on the alignment of the VP1gene sequences of dominant serotypes in environmental surveillance and outbreaks of Shandong and the corresponding strains from GenBank were performed by Mega3.1.【[Results】1. Distribution of HEVs isolated from sewage.From2008to2011,99sewage samples were collected, and72(72.7%) were positive with HEVs. Five type2adenoviruses were also isolated. The HEV isolates contains PV Sabin strains and HEV-B. HEV-B consisted of17serotypes, which were Echo1, Echo3, Echo6-7, Echo11-14, Echo19, Echo25, Echo29-30, CVB1-5. Echo6, Echo7, Echo11, Echo12and CVB3were primary serotypes.2. Variation and recombination analysis of PV isolated from sewage.From the year2008to2011,99sewage samples were collected, and28were PV positive, the positive rate was28.3%.58vaccine-related PV strains were isolated from these sewage samples, none of wild-type poliovirus and vaccine-derived poliovirus was detected.39PV strains were isolated in Jinan city, PV2was the primary serotype. While19PV strains were isolated in Linyi city, PV3was the primary serotype. PV could be found all around the year without seasonality variation. The VP1and3D region of18PV isolated in2010were determined. Homology comparison with Sabin showed that16stains had nucleotide mutation on VP1region, and8strains on3D region. Recombination between serotypes of PV was found in7srains (38.9%), while none of recombination with NPEV was detected.3. Comparison of NPEV distribution between environmental surveillance and patients.Nine NPEV serotypes were detected in the environmental surveillance conducted in Linyi city in2010, which are Echo6-7, Echo11, Echo13, Echo19, Echo30, CBV1-2and CVB4. NPEV strains isolated from patients in the same period were serotyped as Echo6, Echo9, Echol6, Echo30, CVB4, CVA4, CVA9, CVA10, CVA16and EV71. Comparison of distribution showed consistency between environmental surveillance and AFP surveillance.4. Phylogenetic analysis of dominate strains in envionmental surveillance and outbreaks.①CVB3and CVB5are main pathogeny of AM in Shandong province. The genetic relationship among isolates from sewage and AM patients were very approximate due to the low speed of mutation and evolution. More than one transmission chains of CVB3was co-circulating in Shandong province.②Echo6and Echo11less likely to give rise to diseases, and the stains evolution more rapidly compared with CVB3and CVB5. Two lineages were found in environmental surveillance, and different isolates from the same sewage sample were devided into different branches, which indicates several transmission chains was co-circulating at the same time.③Echo30is main pathogeny of AM in Shandong province. The isolation number of this serotype from sewage is small. Isolates from sewage and AM patients had an approximate genetic relationship and were all devided into F genotype.【Conclusion】1. The methods of sewage concentration and virus isolation used in this study is effective in isolating of HEVs from sewage, especially in detecting HEVs that do not cause clinical symptoms. Thus, these methods are the most effective ways to track the non-dominant HEVs infection.2. In the period of maintaining polio-free stage, environmental surveillance, not limited to sample collecting, which can detect PV circulation and variation from external environment samples, is a supplementary way of AFP surveillance system.3. Through homologous comparisons and bioinformatics analysis, variation and recombination of HEVs and genetic evolutionary relationship between stains from surveillance and outbreaks can be obtained.4. Improvement of the methods of concentration and its application in long-term envionmental surveillance are recommended, to investigate the circulation of HEVs and other pathogens and to provide a scientific basis for the prevention of related disease.