海水中胃肠道感染病毒的病原性检测及风险评价
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摘要
胃肠道感染病毒是除细菌之外的引起胃肠道疾病的主要病原体之一，通过接触娱乐用水或食用被污染的贝类感染人体。这类病毒主要来源于人畜粪便等排泄物污染的生活污水，在各种水环境中普遍存在，并且存活时间长，感染剂量低，可以通过娱乐用水或食用海产品感染人类。因此，建立其快速、高效的检测技术，对于水传播疾病的预防和控制是迫切需要的。然而目前尚没有一种同时确定其感染性和快速定量的方法，给人体健康风险评估带来了很大的障碍。因此，本论文从这一点出发，开展了以下相关的工作。
选取具有指示作用的胃肠道感染病毒—肠道病毒（EV）和感染性最强的胃肠道感染病毒—轮状病毒（RV）为指示微生物，实现以下目标：1.建立快速定量水环境中具有感染性病毒浓度的方法，同时探讨不可培养病毒的病原性检测方法；2.将此方法在渤海湾天津海域进行了应用与可行性分析；3.通过探讨病毒在海洋环境中的存活时间和灭活机理，对病毒感染人体的健康风险进行了实际评估。
主要研究内容和研究成果如下：
1.分别建立了肠道病毒代表种脊髓灰质炎病毒（PV）和RV的细胞培养结合实时定量PCR（ICC-qPCR）检测方法，克服了细胞培养和实时定量PCR技术单独作用时的缺点，将细胞培养时间从7天缩短到了2天，并且灵敏度在real-timePCR方法的基础上提高了1个数量级，为细胞病变不明显或水环境中低污染浓度病毒的感染性检测提供了良好的技术支持。
2.在比较超滤和吸附-洗脱这两种水环境中常用病毒浓缩方法的基础上，就不同的浓缩体积、不同孔径滤膜对超滤法浓缩效率的影响进行了探讨。结果表明，超滤浓缩法操作简单、耗时短、对病毒感染性损伤小；在超滤法中，相对于在10L海水中用超滤浓缩装置对病毒进行回收来讲，病毒分别经0.8μm和0.45μm孔径的混合纤维素酯微孔滤膜过滤后，再用超滤管直接对500mL海水中病毒浓缩效果最好。
3.于2010年12月到2011年9月应用ICC-qPCR方法对渤海湾天津近岸海域表层海水中EV和RV进行了为期一年的监测。结果显示，在28个样品中，PV的阳性检出率为57%（16/28），EV71和Coxsackievirus A16为4%（1/28），RV为32%（9/28）。在该海域中，PV的浓度变化范围为0.2～196PFU/L；RV为2～244PFU/L。通过电镜和测序分析鉴定出PV均为Ⅰ型污染，而RV主要是Human rotavirus A的G1血清型污染，其次为G3型。
4.建立了PV和RV的ELISA和特异性RT-PCR检测方法，并将这两种方法进行有机结合，应用到渤海湾表层海水的检测中，试图建立不可培养病毒的病原性检测方法。结果证明该方法能够克服两种手段单独作用时造成的假阳性弊端，并且在很大程度上与ICC-qPCR检测结果一致，适合较高浓度污染的病毒检测，但是由于其灵敏度有限，检测微量病毒污染时可能会造成结果的低估。
5.采取实验室加标PV1疫苗株的方法，选取ICC-qPCR、大片段逐步步移PCR和核酸转染技术，探讨了病毒在自然海水中的存活时间和降解机理。结果显示在实验室不控温、不同盐度条件下，5.0×10~4PFU/mL浓度的PV1在秋冬季最长存活时间为13周，最短为10周。温度和抗病毒微生物是其灭活的主要影响因素，而盐度影响不显著。PV1的3'NCR受到损伤会造成病毒毒力下降，而5'NCR才是决定核酸感染性是否消失的关键。
6.应用评价模型对主要胃肠道感染病毒造成的人体健康风险进行了评估。结果显示，在该研究海域RV相对于PV是主要致病病原体。当游泳者暴露体积为10mL/d时，在该海域感染PV的年风险范围为1.82×10~(-5)（在最低浓度暴露1d）～1.63×10~(-1)（在最高浓度暴露10d），低于可接受年风险（7.0×10~(-3)）的概率为62.5%（暴露1d）～31.25%（暴露10d）；暴露于RV的感染年风险范围为1.17×10~(-2)（在最低浓度暴露1d）～9.93×10~(-1)（在最高浓度暴露10d），远远超过了可接受的年风险。
结论：本论文以PV和RV为试验对象，建立了ICC-qPCR病原性检测方法，为细胞病变不明显或病变时间长的病毒提供了新的检测和分离手段。结合ELISA和特异性RT-PCR方法在很大程度上能够反应不可培养病毒的病原性污染状况，适用于病毒污染比较严重的水体。在本研究设定的条件下，渤海湾表层海水中胃肠道感染病毒浓度，尤其是轮状病毒已经对娱乐者的健康造成了很大的威胁。本研究所建立胃肠道感染病毒的浓缩、检测方法可实现突发性水环境污染事故的快速检测和风险评价。
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 PV1was5.0×10~4PFU/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.
选取具有指示作用的胃肠道感染病毒—肠道病毒（EV）和感染性最强的胃肠道感染病毒—轮状病毒（RV）为指示微生物，实现以下目标：1.建立快速定量水环境中具有感染性病毒浓度的方法，同时探讨不可培养病毒的病原性检测方法；2.将此方法在渤海湾天津海域进行了应用与可行性分析；3.通过探讨病毒在海洋环境中的存活时间和灭活机理，对病毒感染人体的健康风险进行了实际评估。
主要研究内容和研究成果如下：
1.分别建立了肠道病毒代表种脊髓灰质炎病毒（PV）和RV的细胞培养结合实时定量PCR（ICC-qPCR）检测方法，克服了细胞培养和实时定量PCR技术单独作用时的缺点，将细胞培养时间从7天缩短到了2天，并且灵敏度在real-timePCR方法的基础上提高了1个数量级，为细胞病变不明显或水环境中低污染浓度病毒的感染性检测提供了良好的技术支持。
2.在比较超滤和吸附-洗脱这两种水环境中常用病毒浓缩方法的基础上，就不同的浓缩体积、不同孔径滤膜对超滤法浓缩效率的影响进行了探讨。结果表明，超滤浓缩法操作简单、耗时短、对病毒感染性损伤小；在超滤法中，相对于在10L海水中用超滤浓缩装置对病毒进行回收来讲，病毒分别经0.8μm和0.45μm孔径的混合纤维素酯微孔滤膜过滤后，再用超滤管直接对500mL海水中病毒浓缩效果最好。
3.于2010年12月到2011年9月应用ICC-qPCR方法对渤海湾天津近岸海域表层海水中EV和RV进行了为期一年的监测。结果显示，在28个样品中，PV的阳性检出率为57%（16/28），EV71和Coxsackievirus A16为4%（1/28），RV为32%（9/28）。在该海域中，PV的浓度变化范围为0.2～196PFU/L；RV为2～244PFU/L。通过电镜和测序分析鉴定出PV均为Ⅰ型污染，而RV主要是Human rotavirus A的G1血清型污染，其次为G3型。
4.建立了PV和RV的ELISA和特异性RT-PCR检测方法，并将这两种方法进行有机结合，应用到渤海湾表层海水的检测中，试图建立不可培养病毒的病原性检测方法。结果证明该方法能够克服两种手段单独作用时造成的假阳性弊端，并且在很大程度上与ICC-qPCR检测结果一致，适合较高浓度污染的病毒检测，但是由于其灵敏度有限，检测微量病毒污染时可能会造成结果的低估。
5.采取实验室加标PV1疫苗株的方法，选取ICC-qPCR、大片段逐步步移PCR和核酸转染技术，探讨了病毒在自然海水中的存活时间和降解机理。结果显示在实验室不控温、不同盐度条件下，5.0×10~4PFU/mL浓度的PV1在秋冬季最长存活时间为13周，最短为10周。温度和抗病毒微生物是其灭活的主要影响因素，而盐度影响不显著。PV1的3'NCR受到损伤会造成病毒毒力下降，而5'NCR才是决定核酸感染性是否消失的关键。
6.应用评价模型对主要胃肠道感染病毒造成的人体健康风险进行了评估。结果显示，在该研究海域RV相对于PV是主要致病病原体。当游泳者暴露体积为10mL/d时，在该海域感染PV的年风险范围为1.82×10~(-5)（在最低浓度暴露1d）～1.63×10~(-1)（在最高浓度暴露10d），低于可接受年风险（7.0×10~(-3)）的概率为62.5%（暴露1d）～31.25%（暴露10d）；暴露于RV的感染年风险范围为1.17×10~(-2)（在最低浓度暴露1d）～9.93×10~(-1)（在最高浓度暴露10d），远远超过了可接受的年风险。
结论：本论文以PV和RV为试验对象，建立了ICC-qPCR病原性检测方法，为细胞病变不明显或病变时间长的病毒提供了新的检测和分离手段。结合ELISA和特异性RT-PCR方法在很大程度上能够反应不可培养病毒的病原性污染状况，适用于病毒污染比较严重的水体。在本研究设定的条件下，渤海湾表层海水中胃肠道感染病毒浓度，尤其是轮状病毒已经对娱乐者的健康造成了很大的威胁。本研究所建立胃肠道感染病毒的浓缩、检测方法可实现突发性水环境污染事故的快速检测和风险评价。
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 PV1was5.0×10~4PFU/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.
引文
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