水样中病毒浓缩和回收

近年来,由于水污染的加剧,病毒污染引起的水源性疾病正在增多,已经引起人民的广泛关注。即使很少量的病毒颗粒亦可致病。目前各种水样微生物学质量控制主要是通过常规监测粪大肠菌群和大肠埃希菌这样的细菌指示物来实现的,然而大多数观点表明环境水样中细菌和病毒污染水平并不相关,因而建立饮用水中病毒学监测指标十分必要。由于水样中只有少量病毒颗粒存在,进行水样检测前必须浓缩水样中的病毒。水中病毒的检测除了要有准确、灵敏的检测方法外,水中病毒的浓缩与回收的效率亦是决定病毒检测成败的关键。目前水样中病毒浓缩的方法很多,本文对常用的浓缩回收方法作一综述,并对各个浓缩回收方法的优缺点作一评价。     

PCR法检测胰蛋白酶中的猪细小病毒

目的建立原材料胰蛋白酶中猪细小病毒的PCR检测方法。方法提取病毒DNA,PCR扩增目标片段,扩增片段位于猪细小病毒的VP2区,对PCR敏感性进行试验,并应用于原材料胰蛋白酶中的猪细小病毒检测。结果扩增产物在预期位置出现条带,敏感性能检测到100TCID50的病毒量。胰蛋白酶对猪细小病毒检测的灵敏度无抑制作用;3批胰蛋白酶中的猪细小病毒检测结果均为阴性。结论本方法具有良好的敏感性,可用于原材料胰蛋白酶中的猪细小病毒检测。     

贝类海产品中诺如病毒快速检测方法研究

目的：寻找和建立贝类产品中诺如病毒的敏感特异的快速检测方法。方法：通过对报道的3种贝类中病毒提取方法的提取效率进行比较，确定最终的提取方法。设计特异性和敏感性相对较高的套式PCR方法进行病毒检测，检测结果经核酸序列测定证实。结果：3种方法中用PBS-三氯三氟乙烷-PEG处理回收率较高。设计的套式PCR方法可成功检测到病毒，其敏感性可比单轮PCR高100倍。结论：该研究建立了敏感特异的诺如病毒检测方法。     

食品中诺如病毒RT-PCR检测技术研究进展

诺如病毒(Norovirus,NoV)是急性胃肠炎暴发的重要病原,其特殊的生物学特性容易引起食品的污染。对食品中NoV的准确检测能为诺如病毒胃肠炎防控提供科学依据。反转录(RT)-PCR技术是目前食品NoV检测的主要方法,检测过程包括病毒富集、RNA提取、引物设计、RT-PCR反应、扩增产物鉴定共5个步骤,其中病毒富集是检测的关键及难点。影响食品中NoV的RT-PCR检测结果的因素较多,目前尚无一种标准的检验方法,因此有必要加快相关检验方法研究,建立高风险食品的病毒检测方法以指导实际工作。     

048食品中诺如病毒RT-PCR检测技术研究进展

诺如病毒（Norovirus，NoV）是急性胃肠炎暴发的重要病原，其特殊的生物学特性容易引起食品的污染。对食品中NoV的准确检测能为诺如病毒胃肠炎防控提供科学依据。反转录（RT）-PCR技术是目前食品NoV检测的主要方法，检测过程包括病毒富集、RNA提取、引物设计、RT-PCR反应、扩增产物鉴定共5个步骤，其中病毒富集是检测的关键及难点。影响食品中NoV的RT-PCR检测结果的因素较多，目前尚无一种标准的检验方法，因此有必要加快相关检验方法研究，建立高风险食品的病毒检测方法以指导实际工作。     

2011—2015年北京西城区成人门诊腹泻患者诺如病毒与轮状病毒感染的流行病学特征

目的了解门诊腹泻病例中诺如病毒和轮状病毒感染现状,分析本辖区病毒感染性腹泻的流行特征。方法以2011—2015年在北京市西城区两所三甲医院成人肠道门诊就诊的腹泻病例作为监测对象,对其进行诺如病毒和轮状病毒检测及流行病学调查分析。结果调查检测腹泻病例共714例,200例检测阳性,总阳性率28.01%;其中轮状病毒检测阳性率为10.08%;诺如病毒检测阳性率为18.63%。诺如病毒检测阳性率显著高于轮状病毒阳性率(χ~2=7.211,P<0.05)。诺如病毒检测阳性病例中有腹泻10次以上、恶心、呕吐、水样便症状的病例百分比明显高于检测阴性病例;轮状病毒检测阳性病例中有腹痛症状的病例百分比明显低于检测阴性病例,腹泻10次以上的病例百分比明显高于检测阴性病例;诺如病毒及轮状病毒检测阳性病例中,便常规检查分别有白细胞和红细胞的病例百分比均显著低于检测阴性病例,差异有统计学意义。结论北京市西城区成人肠道门诊就诊的腹泻患者中诺如病毒和轮状病毒感染较为普遍,腹泻多次水样便、恶心、呕吐是诺如病毒感染的常见症状;次数较多的无腹痛腹泻是轮状病毒感染的常见表现。     

黄热病病毒检测方法学的研究进展

本文对黄热病病毒检测方法进行了综述。黄热病病毒的检测技术对该病的快速诊断,流行病学调查以及接种人群血清抗体的有效监测意义重大。正确快速地检测出黄热病病毒对保障我国人民健康安全和经济发展均有重大意义。     

SYBR Green Ⅰ实时PCR检测甲肝病毒方法的建立与初步应用

目的建立一种快速、特异的SYBR Green Ⅰ实时PCR方法检测甲肝病毒.方法对SYBR Green Ⅰ实时PCR检测甲肝病毒方法的反应条件、反应体系进行优化,同时与传统RT-PCR方法进行灵敏度比较,提高该方法的特异性、灵敏性.并用该方法对甲肝病毒毒株、脊髓灰质炎病毒、柯萨奇病毒(1～6型)毒株、贝类水产品标本进行检测.结果SYBR Green Ⅰ实时PCR与传统RT-PCR检测甲肝病毒方法的灵敏度无明显区别,但检测时间缩短了1/2.该方法能特异检测出甲肝病毒,不能扩增脊髓灰质炎病毒、柯萨奇病毒(1～6型)毒株,对农贸市场40份贝类水产品进行甲肝病毒核酸检测,检出3份.结论建立的SYBR Green Ⅰ实时PCR方法用于甲肝病毒检测具有特异、灵敏、快速等优点,可用于甲肝的病原学监测.     

贝类中诺如病毒检测方法的研究进展

诺如病毒(norovirus,NVs)是非细菌性胃肠炎的首要致病原因,世界上50%以上的胃肠炎暴发都由它引起。诺如病毒主要通过食物、水以及人与人之间接触传播,被污染的食物中以贝类中富集的诺如病毒最难检测,但贝类在2000—2007年的世界食源性诺如病毒感染中占了17.5%。本文就世界各国对贝类中诺如病毒检测方法的研究进展进行综述。     

SYB GRreenⅠ实时PCR检测甲肝病毒方法的建立与初步应用

目的：建立一种快速、特异的SYBR GreenⅠ实时PCR方法检测甲肝病毒。方法：对SYBR GreenⅠ实时PCR检测甲肝病毒方法的反应条件、反应体系进行优化,同时与传统RT-PCR方法进行灵敏度比较,提高该方法的特异性、灵敏性。并用该方法对甲肝病毒毒株、脊髓灰质炎病毒、柯萨奇病毒（1～6型）毒株、贝类水产品标本进行检测。结果：SYBR GreenⅠ实时PCR与传统RT-PCR检测甲肝病毒方法的灵敏度无明显区别,但检测时间缩短了1/2。该方法能特异检测出甲肝病毒,不能扩增脊髓灰质炎病毒、柯萨奇病毒（1～6型）毒株,对农贸市场40份贝类水产品进行甲肝病毒核酸检测,检出3份。结论：建立的SYBR GreenⅠ实时PCR方法用于甲肝病毒检测具有特异、灵敏、快速等优点,可用于甲肝的病原学监测。 基金 上海市医学重点学科建设项目（05Ⅲ029）     

环境水体中感染性胃肠道病毒检测方法研究进展

胃肠道病毒,是全球范围内引起经水传播的疾病的主要病原物。寻找检测水环境中具有感染性病毒的方法是当务之急。传统细胞培养是鉴定病毒感染力的金标准,但是仍存在费时、费力、成本高且有些病毒（诺如病毒）无法培养的缺点。常用的分子生物学检测方法在检测水中病毒时,虽然有很高的灵敏度和特异性,但亦存在病毒基因拷贝数和感染力之间缺乏相关性的局限性。本文遂对目前国内外在检测感染性病毒的相关研究方面,能部分克服传统细胞培养和直接聚合酶链式反应（PCR）在检测感染性病毒时之局限性的研究成果作一综述报道。     

Evaluation of four cell lines for assay of infectious adenoviruses in water samples

Human viral contamination in drinking and recreational waters poses health risks. The application of PCR-based molecular technology has advanced our knowledge of the occurrence and prevalence of human viruses in water; however, it has provided no information on viral viability and infectivity. Four human cell lines were compared for their sensitivity to different serotypes of human adenoviruses using the TCID₅₀ test. The sensitivity of each cell line varied with different serotypes of adenovirus. Human embryonic kidney cell line 293A and human lung carcinoma cell line A549 were the most sensitive, especially to enteric adenovirus 40 and 41. Plaque assay of primary sewage samples showed 293A can detect viral plaques in 7 of 13 primary sewage samples tested. Adenoviruses were also isolated using 293A from environmental water concentrates. Cloning and sequencing of environmental adenoviral isolates indentified them to be aligned with adenoviruses serotype 40 and serotype 5. The result of this study suggests that plaque assay with 293A cell line is suitable for detection of adenovirus in the aquatic environment. Combining this cell culture with molecular methods for viral assay in the aquatic environment will provide critical information for risk assessment.     

Microcosm environment models for studying the stability of adenovirus and murine norovirus in water and sediment

Microcosms are useful tools for understanding the survival and fate of enteric viruses in aquatic environments. This study set out to determine the stability of infectious enteric viruses in an aquatic environment using a laboratory scale microcosm. Sediment and overlaying water were collected from a lagoon and inoculated with known concentrations of recombinant adenovirus (AdV-GFP) and murine norovirus (MNV-1). Infectious particles of these viruses were measured using fluorescence microscopy (AdV-GFP) or the plaque assay method (MNV-1), over 85 days in two different conditions: under natural sunlight and in fully darkened environments. The time required to reach one log reduction in viral titres (T₉₀) of viable viruses in a natural condition microcosm for AdV-GFP and MNV-1 was shorter than in a dark condition microcosm. There was also a negative correlation between the temperature and infectivity of these viruses in both water and sediment samples. Considering that microcosms aim to mimic natural environment conditions and that AdV-GFP and MNV-1 are excellent surrogates for measuring the infectivity of the respective viruses strains, the results presented here have the potential to be applied in future health hazard studies and also would be useful for future climate scenarios.     

Waterborne human pathogenic viruses of public health concern

In recent years, the impending impact of waterborne pathogens on human health has become a growing concern. Drinking water and recreational exposure to polluted water have shown to be linked to viral infections, since viruses are shed in extremely high numbers in the faeces and vomit of infected individuals and are routinely introduced into the water environment. All of the identified pathogenic viruses that pose a significant public health threat in the water environment are transmitted via the faecal–oral route. This group, are collectively known as enteric viruses, and their possible health effects include gastroenteritis, paralysis, meningitis, hepatitis, respiratory illness and diarrhoea. This review addresses both past and recent investigations into viral contamination of surface waters, with emphasis on six types of potential waterborne human pathogenic viruses. In addition, the viral associated illnesses are outlined with reference to their pathogenesis and routes of transmission.     

Methods to detect infectious human enteric viruses in environmental water samples

Currently, a wide range of analytical methods is available for virus detection in environmental water samples. Molecular methods such as polymerase chain reaction (PCR) and quantitative real time PCR (qPCR) have the highest sensitivity and specificity to investigate virus contamination in water, so they are the most commonly used in environmental virology. Despite great sensitivity of PCR, the main limitation is the lack of the correlation between the detected viral genome and viral infectivity, which limits conclusions regarding the significance for public health. To provide information about the infectivity of the detected viruses, cultivation on animal cell culture is the gold standard. However, cell culture infectivity assays are laborious, time consuming and costly. Also, not all viruses are able to produce cytopathic effect and viruses such as human noroviruses have no available cell line for propagation. In this brief review, we present a summary and critical evaluation of different approaches that have been recently proposed to overcome limitations of the traditional cell culture assay and PCR assay such as integrated cell culture-PCR, detection of genome integrity, detection of capsid integrity, and measurement of oxidative damages on viral capsid protein. Techniques for rapid detection of infectious viruses such as fluorescence microscopy and automated flow cytometry have also been suggested to assess virus infectivity in water samples.     

Epidemiology and detection as options for control of viral and parasitic foodborne disease.

Human enteric viruses and protozoal parasites are important causes of emerging food and waterborne disease. Epidemiologic investigation and detection of the agents in clinical, food, and water specimens, which are traditionally used to establish the cause of disease outbreaks, are either cumbersome, expensive, and frequently unavailable or unattempted for the important food and waterborne enteric viruses and protozoa. However, the recent introduction of regulatory testing mandates, alternative testing strategies, and increased epidemiologic surveillance for food and waterborne disease should significantly improve the ability to detect and control these agents. We discuss new methods of investigating foodborne viral and parasitic disease and the future of these methods in recognizing, identifying, and controlling disease agents.     

Effects on health of water and food contamination by emergent human viruses

The development of molecular technologies applied to environmental studies has shown that even in highly industrialized countries there is a high prevalence of viruses in the environment that represents an important impact on public health and substantial economic losses mainly related to the transmission of viruses through water and food. Significant concentrations of viruses are detected in the water flowed to the environment and in the biosolids generated in wastewater treatment plants. This work describes the general characteristics of the environmental contamination by viruses principally by emergent viruses, with a special emphasis on the hepatitis E virus (HEV) and the human polyomaviruses as the environmental contaminants more recently identified in industrialized countries. It has been shown that there is a high prevalence of the human polyomaviruses BKV and JCV in urban sewage in all studied countries, implying a potential transmission of these viruses and their potential oncogenic genes through the oral route. Recent studies have shown that the epidemiological pattern of the HEV infection in industrialized countries is complex and that a diversity of HEV strains simultaneously infects the population. The control of the viral contamination requires the standardization of molecular techniques and the development of a surveillance program for the evaluation of the viral parameters and to reduce the dissemination of already established diseases and emergent viral infections.     

Characterisation of a diverse range of circular replication-associated protein encoding DNA viruses recovered from a sewage treatment oxidation pond

Our knowledge of circular replication-associated protein encoding single-stranded (CRESS) DNA virus diversity has increased dramatically in recent years, largely due to advances in high-throughput sequencing technologies. These viruses are apparently major virome components in most terrestrial and aquatic environments and it is therefore of interest to determine their diversity at the interfaces between these environments. Treated sewage water is a particularly interesting interface between terrestrial and aquatic viromes in that it is directly pumped into waterways and is likely to contain virus populations that have been strongly impacted by humans. We used a combination of high-throughput sequencing, full genome PCR amplification, cloning and Sanger sequencing to investigate the diversity of CRESS DNA viruses present in a sewage oxidation pond. Using this approach, we recovered 50 putatively complete novel CRESS viral genomes (it remains possible that some are components of multipartite viral genomes) and 11 putatively sub-genome-length circular DNA molecules which may be either defective genomes or components of multipartite genomes. Thirteen of the genomes have bidirectional genome organisations and share similar conserved replication-associated protein (Rep) motifs to those of the gemycircularviruses: a group that in turn is most closely related to the geminiviruses. The remaining 37 viral genomes share very low degrees of Rep similarity to those of all other known CRESS DNA viruses. This number of highly divergent CRESS DNA virus genomes within a single sewage treatment pond further reinforces the notion that there likely exist hundreds of completely unknown genus/family level CRESS DNA virus groupings.     

Detection of airborne viruses in a pediatrics department measured using real-time qPCR coupled to an air-sampling filter method

Children are vulnerable to viral infections. The study discussed in this article investigates the possibility of aerosol transmission of viruses in children under age 18 in the pediatrics department of a medical center in Taipei, Taiwan. After first using the filtration method to collect viral aerosols, the authors combined it with real-time quantitative polymerase chain reaction (qPCR) to detect influenza A virus (INFAV), human adenovirus (HAdV), and enterovirus. Of 33 aerosol samples collected from the emergency room of the pediatrics department, 8 (24%) were positive for INFAV, 12 (36%) for HAdV, and 5 (15%) for enterovirus. HAdV was detected from the aerosol of 26 samples, but INFAV and enteroviruses were not. The filter and real-time qPCR can be used to detect and quantify the viral load in aerosols, in which enteroviruses had the highest viral titer. In summary, a well-established filter/real-time qPCR assay was successfully applied to measure viral aerosols in the occupational environment. Environmental monitoring of airborne viruses could provide an early indicator of dangerous viruses in the air of hospitals.     

微流体功能芯片检测装置在流感病毒检测中的初步研究

目的 建立一种新型有效、快速、灵敏的呼吸道病毒检测技术。方法 用微流体生物功能芯片微量检测系统检测传染性强、潜在世界大流行危害的流感病毒。结果 该技术检测限度约在病毒粒子10^4。结论 初步研究表明微流体生物功能芯片是一种快速、灵敏的检测系统,对病原微生物感染,尤其是对经空气传播的病毒的快速检测辅助临床诊断具有重要作用。此外在控制病房的院内感染也将发挥其作用。