The Characteristic Analysis Of Occurrences And Distribution Of Typical Viruses In Domestic Sewage And Reclaimed Wastewater

Various viruses exist in wastewater. Different viruses and even the viruses belonging to the same species but different types possess different pathogenicity and resistance; thereby the health risks caused by these viruses vary a lot. Moreover, the source of the reclaimed wastewater is raw sewage, when the raw sewage was treated insufficiently or the reclaimed wastewater was stored improperly, the exposure risk for human beings would be increased by using such reclaimed wastewater. However, so far there are no specific and unified standards for detection and evaluation of viral contamination in wastewater. Thus, comprehensive investigation on understanding the characteristics of occurrence and genotype distribution of viruses in wastewater and reclaimed wastewater is of great significance for prevention and control of viral contamination, guaranteeing the water quality security and decreasing the human health risk.The polymerase chain reaction(PCR) based on nucleic acid detection and its derived methods showed the advantages of short time for detection, high sensitivity, and high specificity and so on, they have been extensively used in environmental virological study, thus these advanced methods would also be applied in our research for viral detection in order to rapid analyze the source, genotype distribution and variation of viruses in the tested samples.This study is part of the project of National Natural Science Foundation of China. From the viewpoint of hygiene security of water quality, the study aims to analyze the viral contamination in wastewater and reclaimed wastewater. Enterovirus(including the hand-foot-and-mouth diseases viruses, HFMD viruses) and some gastroenteritis viruses are selected as typical viral contaminants in wastewater. And the viral concentration method suitable for environmental water samples, the PCR-DGGE method for genotyping detection of enteroviruses, the molecular assays for analyzing the distribution of typical gastroenteritis viruses and the HFMD viral pathogens were established respectively. The established methods were applied to detection of viruses in wastewater and reclaimed wastewater, and the methods were proved to be effective. For the wastewater treatment and reclamation process, the occurrences, genotype distribution and variation of typical viruses were analyzed. Meanwhile, the variation characteristics of viral nucleic acid and infectivity were analyzed respectively by enterovirus tracer experiment. The main work and achievements are as follows:(1) To establish the proper viral concentration method for environmental water samples, the suitability of three different viral concentration methods was analyzed. After comparison of the impact of different types of wastewater samples and viruses on the viral recovery efficiency, it showed that the PEG precipitation method showed higher viral recovery efficiency for both raw sewage and treated wastewater samples in which the suspended solid was higher. The membrane adsorption and elution method was proved to be more suitable for viral concentration of lake water with low turbidity, but in order to improve the detection sensitivity, the membrane adsorption and elution method followed by PEG precipitation as a second concentration method would be better. SiO2 adsorption method showed low viral recoveries for all three types of wastewater samples.(2) The PCR-DGGE method for detection of enteroviruses and the(semi)nested-PCR assays for detection of typical gastroenteritis viruses were established respectively to understand the occurrence and distribution of the selected typical viruses in wastewater. The established methods were applied on investigating the viral contamination in wastewater treatment and reclamation system, the results showed that universal primer set A targeting the middle region of enterovirus 5’UTR for PCR amplification and 10% polyacrylamide gel with the denaturant concentration ranging from 10% to 40% for DGGE could effectively separate the characteristic bands representing for different types of enteroviruses. By comparison of the detection results between enteroviruses and typical gastroenteritis viruses, it showed that the sources of viruses in wastewater were extensive, the viral contamination could be decreased to some extent in the A2 O biological wastewater treatment unit, and viruses could be significantly removed in the following MBR process, finally, the viral pathogens would be inactivated by sufficient chlorination. However, the viruses were detected again when the disinfected reclaimed wastewater was stored in the artificial lakes, it might indicate that there was unidentified non-point contamination source. The detection of typical gastroenteritis viruses could be affected by sampling time, area and infected population. Meanwhile, the occurrence of enteroviruses could be more stable and frequent, and PV1 could be existed stably in all the wastewater samples except the disinfected MBR sample, and it might indicate that enterovirus may be the potential viral indicator to indicate the viral contamination in the system.(3) Three enteroviruses(EV71, CVA16 and CVA10) which could frequently cause HFMD were selected, the PCR assay for analysis of the three major HFMD viral pathogens was developed, and the established method was applied on assessing the occurrence and distribution of the three major HFMD viruses in domestic wastewater, the relationship between HFMD viruses and enterovirus, HFMD viruses and conventional water quality index were also analyzed. A semi-nested PCR primer set was designed targeting the nucleotide region from 5’UTR to VP2 of the three viruses, the semi-nested PCR assay was established after optimization of the PCR condition. According to the results of long-term monitoring of the raw sewage and treated wastewater in WWTP using the established method, it showed that the occurrences of the three major HFMD viruses was much higher from April to July when HFMD was prevalent; CVA10 was the major HFMD viral pathogen in the samples. By comparison with the qualitative detection of enteroviruses, it showed that the occurrences of pan-enteroviruses would not be varied with that of HFMD viruses. But there seemed to be a similar trend between the occurrences of HFMD viruses and the variation of the conventional water quality indicators of the raw sewage, and the occurrences of HFMD viruses showed positive correlated with SS.(4) The experiments using enterovirus tracer(poliovirus) were conducted to investigate the variation of viral nucleic acid and infectivity during different stage in wastewater treatment process. The results showed that: There might be differences between variation of viral nucleic acid and infectivity during different treatment stage; Inorganic particles such as Kaolin could lead to the decrease of both viral nucleic acid and infectivity, and the viral reduction rate was about 0.8~0.9-log; The reduction rate of viral infectivity(~1.7-log) was higher than that of the nucleic acid(~1.2-log) during the activated sludge process, and the microorganisms in activated sludge might result in the rapid decrease of viral activity; The viral removal by membrane filtration was obviously influenced by the membrane pore size, and as for the same pore size, the removal of infectivity might be equivalent to that of the nucleic acid; The viral removal by sodium hypochlorite and ultraviolet was dependent on the Ct value(time or dose dependent), and the viral nucleic acid disappeared more quickly than the infectivity under the condition of a certain Ct values; The low temperature was preferable for viral survival under natural condition, and the reduction of viral infectivity was lower than that of the nucleic acid.