肠道病毒在水环境中的分布及其行为

肠道病毒都是通过粪便排出体外的,因此它们很容易进入污水和各种天然水体中。从粪便排出的病毒有100多个型别。每克粪便中可含100多万个病毒颗粒,已经从污水,地表水,地下水和海水中检出了许多型别的肠道病毒,有些肠道病毒可在水环境中存活很长时间,已报道的病毒存活时间范围是:自来水2—168天;海水2—130天;河水2—168天;土壤25—125天;牡蛎6—90天,海水沉积物8—20天。水环境中的病毒存活受很多因素的影响,包括温度,日光,吸附作用和生物因素,在自然状态下,病毒存在有三种形式:单个病毒,病毒彼此聚集;吸附或埋入固体物中。病毒的存在形式对病毒存活有很大影响。由于人为的和自然的活动,使水环境中的病毒迁移和传播的途径较多,从而使病毒再次从自然界回到人体,保持平衡。     

环境介质中病毒生态的研究

病毒是许多人及重要经济动、植物病患的病原。一些病毒在环境中可因条件不同而生存数小时到数月, 并在水、气、士中迁移达若干公里。现有的污水处理方法对病毒, 特别是肠道病毒效果欠佳, 土地处置原污泥以及污水灌溉的水果和蔬菜能传播人肠道病毒。即使小至一个组织培养的感染剂量(病毒)也可引起人的疾病, 因此对环境介质中, 特别是饮水和食物中的少量病毒的去除也是重要的。现有的指示物不能确切地指示粪便污染, 更不能充分反映人肠道病毒的污染。大肠菌噬菌体在地表水、地下水和污水中比人肠道病毒更呈持久性, 还有许多适于选择分析技术特有性能, 因此很可能在一定条件下用它作人肠道病毒的指示物。作者对我国今后需要开展的研究提出了建议。     

人胎肾和人胎肺细胞对肠道病毒的易感性

猴肾细咆和人羊膜细胞虽然对大多数类型的肠道病毒易感,但由于猴肾细胞来源有限和人羊膜细胞制备的成功率较低,昕以需要寻求其他更合适的细胞来代替。为了确定人胎肾和人胎啼细胞在肠道病毒工作中的使用价值,本文较系统地比较了这两种细胞对晒遒病毒的易感性。实验证明,除了Co～ckie B2和H4型病毒,ECHO 9、18、20和2 7型病毒在人胎肾细胞及ECHO、22和23型病毒在人胎肺细胞的滴度不够理想外,这两种细胞对所研究的其余类型的病毒都较易感。大多数类型的肠道病毒不但能在人胎肾和人胎肺细胞中培养和传代,而且原有皿凝者仍能保持其血凝特性。用猴肾、人胎肾和人胎肺细胞从122份粪便标本分离病毒,结果共有64份标本阳性。其中用猴肾细胞分离得到阳性结果的有;4份,用人胎肾细胞有56份,用人胎肺细胞有51份。猴肾细胞对脊髓灰质炎病毒比人胎肾和人胎肺细胞易感,而人胎肾和人胎肺细胞对某些非脊髓灰质炎病毒比猴肾细胞易感。实验结果表明,在肠道病毒的工作中可以应用这两种细胞培养来代替猴肾细胞培养。     

高碑店污水系统病毒污染情况的检测

本文报道用石英粉吸附——洗脱法浓集污水病毒,其回收率为51～90％。对1985年3～7月采集的不同水样进行病毒分离和蚀斑滴定,结果表明,高碑店污水系统中的病毒大部分为肠道病毒。春季以脊髓灰质炎病毒为主,夏季以柯赛奇B3及非肠道病毒为主。不同类型污水中的病毒浓度依次为:原污水58PFU/1;二级污水<19,约为9PFU/1;医用污水<16,约为1PFU/1,地下水<1.7PFU/1,未检出病毒。夏季水样内病毒浓度较春季低。     

医院污水中脊髓灰质炎等肠道病毒消长动态的初步观察

在废水中有100种以上对人有致病性的不同类型肠道病毒,浓度可达到10,000～100,000PFU/升,并且有在水中存活数月的能力。因此加强水中病毒的监测,并根据不同季节病毒存在动态给予严格地消毒措施,是控制疾病流行的有力保证。     

2011—2014年百色市重症手足口病流行病学及病原学分析

目的了解2011—2014年百色市重症手足口病的流行病学及病原学特点,为预防和控制重症手足口病提供依据。方法收集2011—2014年百色市重症手足口病疫情资料,进行描述性流行病学分析;采用实时荧光定量PCR方法对手足口病重症病例标本进行肠道病毒通用型、肠道病毒71型和柯萨奇病毒A16型核酸检测。结果2011—2014年百色市共报告手足口病105 900例,其中重症手足口病537例,重症率为0.51%;死亡35例,病死率为0.03%。重症手足口病发病主要集中在每年的4—5月;3岁以下重症病例占重症病例总数的88.27%,男女性别比为1.44∶1;散居儿童是主要的发病人群(75.98%);患者主要集中在平果县、田东县、田阳县、右江区等右江河谷一带;重症手足口病阳性患者中,肠道病毒71型占77.51%,柯萨奇病毒A16型占5.15%,其他肠道病毒占17.34%。结论 2011—2014年百色市重症手足口病多发于3岁以下的散居儿童,发病季节主要集中在4—5月,病原体以肠道病毒71型为主。     

GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒—人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型。优化多重反应体系中针对5’UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA。该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查。     

不同温度条件下霍乱弧菌生存能力的实验研究

目的通过在不同温度条件下对霍乱弧菌生存能力的观察,探索大连市水域中霍乱弧菌在流行＂间歇期＂如何存活和越冬。方法在自然海水和湖水中分别接种本市分离出的埃尔托型霍乱弧菌流行株小川1b型（大E940014）和埃尔托型霍乱弧菌非流行株小川32l（大E940011）,在1～2℃、5～6℃及10～12℃3个温度条件下检测霍乱弧菌生存能力。结果流行株（小川1b型）在1～2℃海水存活26 d,5～6℃存活28 d,10～12℃存活46 d。可见,10～12℃生存时间明显高于1～2℃或5～6℃（P〈0.05）。结论两类菌株的生存对环境温度均很敏感,温度越低死亡越快。在自然水体中,非流行株生存时间略长于流行株;而在除菌水体中两类菌存活能力基本一致。     

一起传染病暴发中肠道病毒血清型鉴定和ECHO30基因特征分析

2003年5～9月,山东省泰安市发生了由肠道病毒(Enterovirus,EV)感染所致的传染病暴发,临床症状以手足口病(HFMD)为主,同时有心肌炎和无菌性脑膜炎等中枢神经系统症状患者也占较大比例。131份病人(粪便、咽拭子、脑脊液)标本中共分离到EV62株,其中ECHO1939株,EV716株,ECHO304株,其它肠道病毒13株。4株ECHO30病毒中的2株分离自2个患者的粪便标本,但用WHO肠道组合血清中和试验未能定出型别。另外2株分离自同一患者的粪便和脑脊液标本。病原学分析表明,ECHO30是引起该患者无菌性脑膜炎的病原。抗E—CHO30标准株的血清中和这4株病毒的滴度低于标准株5～20倍。VP1区全基因序列测定和同源性比较分析表明,4株ECHO30分离株病毒核苷酸同源性在98．0％～98．5％,氨基酸同源性在98．9％～99．3％,提示这4株病毒来源于同一传播链,2003年5～9月ECHO30在该地区可能有局部流行。系统进化树分析表明,ECHO30病毒可以划分为6个基因型,其中基因型1～5为GenBank中已发表的ECHO30分离株,山东分离株与其它5个基因型成员核苷酸差异分别在9．4％～24．4％,在进化树上形成了较独立的分支,是一个新基因型,将其划分为第6基因型。     

An oligonucleotide hybridization assay for the identification and enumeration of F-specific RNA phages in surface water

F-specific RNA phages can be used as model organisms for enteric viruses to monitor the effectiveness of sewage treatment, and to assess the potential contamination of surface water with these viruses. In this paper a method is described which identifies RNA phages quantitatively by a plaque hybridization assay. Oligonucleotide probes were developed that can assign phages to their phylogenetic subgroups. Such a distinction is important, since some subgroups preferentially occur in sewage of human origin, while others tend to be associated with animal wastewater. The method has been tested on a large number of isolates and represents an improvement in time and reliability over the previously used serological classification.     

Detection and molecular characterization of enteric viruses in environmental samples in Monastir,Tunisia between January 2003 and April 2007

Aims: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. Methods and Results: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). Conclusion: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. Significance and Impact of the Study: Our results address the potential health risks associated with transmission of human enteric viruses through water‐related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.     

Human pathogenic viruses at sewage sludge disposal sites in the Middle Atlantic region.

Human enteric viruses were detected in samples of water, crabs, and bottom sediments obtained from two sewage sludge disposal sites in the Atlantic Ocean. Viruses were isolated from sediments 17 months after the cessation of sludge dumping. These findings indicate that, under natural conditions, viruses can survive for a long period of time in the marine environment and that they may present potential public health problems to humans using these resources for food and recreation. The isolation of viruses in the absence of fecal indicator bacteria reinforces previous observations on the inadequacy of these bacteria for predicting the virological quality of water and shellfish.     

Human pathogenic viruses at sewage sludge disposal sites in the Middle Atlantic region

Human enteric viruses were detected in samples of water, crabs, and bottom sediments obtained from two sewage sludge disposal sites in the Atlantic Ocean. Viruses were isolated from sediments 17 months after the cessation of sludge dumping. These findings indicate that, under natural conditions, viruses can survive for a long period of time in the marine environment and that they may present potential public health problems to humans using these resources for food and recreation. The isolation of viruses in the absence of fecal indicator bacteria reinforces previous observations on the inadequacy of these bacteria for predicting the virological quality of water and shellfish.     

Environmental surveillance and molecular characterization of human enteric viruses in tropical urban wastewaters

Aims: To study the prevalence and genotypes of waterborne pathogenic viruses in urban wastewaters in the tropical region. Methods and Results: Viruses in wastewaters collected at three water reclamation plants in Singapore were studied by molecular methods. Over a 6‐month sampling period, adenoviruses, astroviruses and both norovirus genogroups I (GI) and II (GII) were detected in 100% of the sewage and secondary effluent. Enteroviruses and hepatitis A viruses (HAV) were found in 94 and 78% of sewage, and 89 and 28% of secondary effluent, respectively. By using quantitative real‐time PCR, estimated concentrations of astrovirus in the sewage were 1–2 orders of magnitude higher than those for adenovirus, noroviruses GI and GII. Genotyping of environmental isolates revealed multiple genotypes of GI and GII noroviruses. Coxsackieviruses A, astrovirus type 1 and adenovirus type 41 were prevalent. Norovirus GII/4 and coxsackievirus A24 isolates in wastewaters were closely related to respective outbreak strains isolated previously in Singapore. Conclusions: This study showed the widespread occurrence of all tested enteric virus groups in urban wastewaters. Genetic diversity of astroviruses, enteroviruses and noroviruses in the tropical region was observed. Significance and Impact of the Study: The high prevalence and great genetic diversity of human enteric viruses in urban wastewaters strongly supports the need of further comprehensive studies for evaluating the public health risk associated with viral pathogens in water environments.     

Sewage bacteriophage photoinactivation by cationic porphyrins: a study of charge effect.

Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Recently the inactivation of bacteria in residual waters has been reported, but nothing is known about photoinactivation of environmental bacteriophages, which are often used as indicators of human enteric viruses. In this study we tested the effect of six cationic porphyrin derivatives with two to four charges on the photoinactivation of a sewage bacteriophage. A phage suspension of 5 x 10(7) PFU mL(-1) was exposed to white light (40 W m(-2)), during 270 min, at three photosensitizer concentrations (0.5, 1.0 and 5.0 microM). Tetra- and tricationic porphyrins inactivated the T4-like sewage phage to the limits of detection, but dicationic porphyrins did not lead to a significant decrease in phage viability. At the highest photosensitizer concentration (5.0 microM), the phage was completely inactivated (>99.9999% of inactivation, reduction of 7.2 log) after 270 min by the tetracationic porphyrin. Two of the tricationic derivatives also led to phage inactivation to the limit of detection. The rate of bacteriophage photoinactivation and the efficiency of the photosensitizer appeared to vary with the charge and with the substituents in the meso-positions of the porphyrin macrocycle. Tetra- and tricationic porphyrins can, therefore, be used as a new method for inactivating sewage bacteriophages that are frequently used as human enteric virus indicators. The complete inactivation of viruses with low light intensity means that this methodology can be used even on cloudy days and during winter, opening the possibility to develop new technologies for wastewater treatment.     

Human enteric viruses–potential indicators for enhanced monitoring of recreational water quality

Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O‘ahu, Hawai‘i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s12250-015-3644-x and is accessible for authorized users.     

Virological quality of the Ria de Aveiro: Validity of potential microbial indicators

The summer occurrence of human enteroviruses and rotaviruses in the bacteriologically clean area of the Ria de Aveiro, a coastal marine lagoon, prompted the question of the assessment of the virological quality of recreational waters and shellfish raising beds. Enteroviruses were present in surface water at a density of 3 pfu 10 l^–1 and were accumulated in sediments and, especially, in cockles where they reached concentrations 2 to 3¹⁰log units greater. Rotaviruses were detected at one¹⁰log unit below the density of enteroviruses in sediments and cockles and were not detected in water. Four bacteriophage systems were assayed as indicators of human enteric viruses: somatic coliphages ofE.coli C, sexual and sexual-RNA coliphages plated onSalmonella WG49 and phages againstBacteroides fragilis HSP40. The results obtained from 2 lagoon stations sampled in summer, autumn and winter showed that the four systems failed to indicate the presence of enteroviruses and rotaviruses in water, sediment and shellfish samples. The absence of phages ofB. fragilis HSP40 in all types of samples taken from the lagoon, but not from the residual waters of the treatment station, suggests that they may suffer a strong negative pressure in this ecosystem as their proportion to the coliphages in the cockles deviated strongly from the ratio of 1100 to 11000 observed at the sewage outfall. In fact, no correlation was observed between these phages and enteric viruses or coliphages. Alternatively, it is possible that the importance of diffuse faecal pollution and the interference of faecal pollutants of animal origin, including migratory sea birds which are abundant in winter, can alter the proportions of the faecal bacteriophages beyond recognition. It is apparent that bacteriophage monitoring of the health risk linked to the occurrence of viruses in the marine environment is not yet fully resolved, what may leave viral quality assessment dependent on direct detection of human enteric virus.