The Pathogenic Detection Of Enteric Viruses In Seawater And Human Health Risk Assessment

Enteric viruses are one of the main nonbacterial agents of gastroenteritis, whichtransmit through contaminated recreational water and shellfish to human. Theymainly derive from sewage which are polluted by human and animal feces, and havefrequently been detected in various types of environmental waters. Due to their longduration and low dose-response, they can cause the infection of human principallythrough the polluted recreational water and shellfish. Thus, it is very important toestablish a rapid and efficient method for the quantitation of this kind of virus inenvironmental water to prevent and control their prevalence. However，there is stillno one method which not only can ensure their infectivities but quantify them rapidly,which makes a lot of obstacles to assess their risk to human health. Therefore, wecarried out the following works.The index of enteric viruses-enterovirus （EV） and the most infective agent in allthe enteric viruses-rotavirus （RV） were selected as indicators to achieve these objects:First, establish the method to quantify the concentration of infectious enteric viruses,and the technology about the pathogenic detection of non-cultural virus was alsoexplored. Second, the established methods were applied in Bohai Bay, Tianjin tostudy its feasibility. Third, the mechanism of the inactivated rate and survival time ofenterovirus in the natural marine environment was described as well. Based on thisexperimental data, the infected risks of enterovirus and rotavirus were estimated.The main contents and results are as follows:1. Cell culture integrating real-time quantitative polymerase chain reaction（ICC-qPCR） of representative of poliovirus （PV） and RV were established,respectively. Compared with cell culture and real-time PCR assays independentlyICC-qPCR has overcomed their disadvantages, which can shorten the viral detectiontime from seven days by the traditional cell culture to two days, and improve thesensitivity for1-fold on the basis of real-time PCR technology. The other advantageof ICC-qPCR is being able to provide technical support for quantifying virons that are unambiguously infectious, a particular advantage for infectious viruses that have lowdensities.2. Based on the comparison of two common viral recovery methods in watersamples, namely ultrafiltration and adsorption-elution, the effects of two factorsincluding volume and nominal pore size were discussed to optimize ultrafiltrationmethod. Results presented that ultrafiltration operated simply, took short time as wellas little damage to the viral pathogenicity; Compared with recovery from10-Lvolume, virus directly ultra-filtrated from500-mL seawater sample by CentrifugalFilter Device after using the0.8-μm and0.45-μm mixed ester millipore filter was themost efficient concentration method.3. In this work, ICC-qPCR method were employed, twenty-eight surfaceseawater samples were collected from12,2010to9,2011in Bohai Bay, Tianjin,China. The results showed that57%,4%,4%,32%samples were positive forinfectious poliovirus, enterovirus71, Coxsackievirus A16and rotavirus, respectively.Concentrations of infectious poliovirus and rotavirus in this coast samples weredetermined to be0.2～196PFU/L and2～244PFU/L. All the poliovirus sampleswere characterized asⅠ serotype usingelectron microscope observation andsequencing analysis, and the pollution of rotavirus mainly were human rotavirus A G1serotype, followed by G3type.4. ELISA method and specific RT-PCR were established and combined to detectPV and RV in the surface seawater of Bohai Bay. We aimed at establishing thedetection method of viral infectivity for non-cultural types. The result demonstratedthat this method can overcome the false-positive disadvantage of two separated roles,which can obtain consistent results with ICC-qPCR test to a great extent, and suit fordetecting the viral pollution with relatively high level. But the risk may beunderestimated due to its limited sensitivity when the density of virus was generallyin low range.5. PV1Sabin strains were used as a model to study the survival and degradatingmechanism of virus in natural seawater. ICC-qPCR, Long-overlapping PCR andRNA transfection technologies were employed. The results showed that when theconcentration of PV1was5.0×10⁴PFU/mL, they can survival for10to13weeks under the laboratory temperature in autumn-winter at different salinity. Compared tosalinity, the temperature is the main factor of virus die-off. When PV13’NCR wasdamaged the viral virulence will decrease, while5’ NCR is the key factor to the viralinfectivity.6. Quantitative models of risk assessment were employed to estimate the humanhealth risk when exposed to the main enteric viruses. The results demonstrated thatcompared with poliovirus, rotavirus is the major pathogenic pathogen in this coast.The estimated annual risk of probability caused by poliovirus during humanswimming in this coastal area was found to be1.82×10^-5（1day exposure to thelowest concentration）～1.63×10^-1（10days exposure to the highest concentration）.The probability conforming to the water safe associated PV was62.5%（10daysexposure）～31.25%（10days exposure）；While exposed to RV, the risk ranged from1.17×10^-2（1day exposure to the lowest concentration） to9.93×10^-1（10daysexposure to the highest concentration）, the hazards have far exceeded the acceptablethreshold.Conclusions: ICC-qPCR assays were established to detect the infectious EV andRV, the primary advantage of this method is to quantify virions that areunambiguously infectious, a particular advantage for viruses that do not produce CPE.Combined ELISA and specific RT-PCR methods can reflect the pathogenic pollutionof unculturable viruses to large extents, which is suitable for the water environmentthat the concentration of virus is relatively high. The concentration of enteric virusesin the surface seawater of Bohai Bay may cause great health threaten to the health ofrecreationer, especially rotavirus. The assay of viral recovery and detectionestablished in this work can realize the fast detection and risk assessment when thewater emergent contaminated events occur.