key: cord-266670-jxgywvwx
authors: Wong, Mark; Xagoraraki, Irene; Rose, Joan B.
title: Chapter 13 Recent Advances and Future Needs in Environmental Virology
date: 2007-09-06
journal: Perspect Med Virol
DOI: 10.1016/s0168-7069(07)17013-0
sha: 
doc_id: 266670
cord_uid: jxgywvwx

The detection of viruses in water and other environmental samples constitutes special challenges. The standard method of detection of viral pathogens in environmental samples uses assays in mammalian cell culture. The infected cell cultures undergo observable morphological changes called cytopathogenic effects (CPEs) that are used for the detection of viruses. Even though many viruses are culturable in several cell lines and are thus detectable by the development of CPEs in cell culture, there are several viruses, like enteric waterborne adenoviruses types 40 and 41, which are difficult to culture and do not produce clear and consistent CPE. Other viruses, like waterborne caliciviruses, have not yet been successfully grown in cell cultures. Conventional cell culture assays for the detection of viruses in environmental samples have limited sensitivity and can be labor-intensive and timeconsuming. Two advances, the PCR and microarrays, have spurred the study of viruses and should be further applied to the field of environmental virology. The ability of both DNA viruses and RNA viruses to rapidly evolve means new and emerging viral pathogens will need to be addressed. Pathogen discovery and characterization, occurrence in the environment, exposure pathways, and health outcomes via environmental exposure need to be addressed. This will likely follow a new microbial risk framework that will require focused research on some important properties of viral disease transmission. The future will require models that examine community risks and provide explicit links between the models currently under development for environmental exposure and infectious disease.

The global outbreaks of severe acute respiratory syndrome (SARS) and avian influenza (AI) highlight the degree of vulnerability that high-density urban populations face when threatened by novel, unanticipated viral pathogens. This is further underscored by security fears brought on by recent acts of terrorism both in the US and abroad. Less sensational but equally serious outbreaks of many other viruses like norovirus, hantavirus, and West Nile virus have been documented worldwide and are on the rise. Rotavirus-induced diarrhea is still the most prevalent infant killer in many developing nations causing an estimated 140 million cases worldwide and killing almost 600,000 people annually (Parashar et al., 2003) .

A United Nations report on the world water crisis situation has highlighted the twin issues of scarcity and impacted quality of the world's drinking water supply (UNESCO, 2003) . Scarcity of water in many parts of the world has forced people to turn to increasingly less pristine sources of water for their drinking and other needs. This has given rise to an increased incidence of waterborne disease. The burgeoning world population is also placing a greater strain on the current world water supply. Irrigation currently consumes approximately 70-80% of the world's fresh and groundwater supply while polluting lakes and streams through surface runoff of pesticides, fertilizers, and land-applied biosolids with their associated pathogen loads. The US Food and Agricultural Organization (FAO) anticipates a net expansion of irrigated land of some 45 million hectares in 93 developing countries to a total of 242 million hectares in 2030 and projects that agricultural-water withdrawals will increase by some 14% from 2000 to 2030 to meet future food production needs (FAO, 2002) .

The increase in population also means that more wastewater is being generated. Globally, 41% of the population is without adequate sanitation and very little of the global wastewater is treated. (WHO/WSH main site: www.who.int/water_sanitation_ health/). According to the US House Transportation Subcommittee on Water Resources (2003) , after investing $250 billion in wastewater infrastructure in the US with the passage of the Clean Water Act of 1972, our communities' economic wellbeing, which relies on clean water, and our ability to continue to meet the public health goals are at risk. This is despite there being 16,000 publicly owned wastewater treatment plants, 100,000 major pumping stations, 600,000 miles of sanitary sewers, and 200,000 miles of storm sewers in the US.

Historically, the importance of protecting one's drinking water supply has been well documented and recognized. Poisoning or contaminating an enemies' water supply has been practiced in warfare since at least the fourth century BC. Less deliberate acts of contamination occur more frequently due to industrial accidents, inclement weather, and weak enforcement of regulations or operator neglect. While animal wastes have been implicated in bacterial and parasitic outbreaks, the viruses remain associated with some of the most serious health consequences such as the outbreak of viral hepatitis E in Kanpur, India in 1991 that affected an estimated 79,091 people (Naik et al., 1992) with 30% mortality in pregnant women in the first trimester. The recent widespread poliovirus outbreaks throughout Africa are likely in part due to contaminated water and the inadequate and wastewater treatment (Pavlov et al., 2005) .

More recently, attention has also focused on the need to protect recreational water sources (Wade et al., 2003; Standish-Lee and Loboschefsky, 2006) . Fresh and salt water sources represent an important recreational resource, especially to economies that rely heavily on tourism. In addition, the increasing scarcity of pristine water sources has meant that the water cycle is being short circuited in order to provide adequate water for drinking, recreation, power generation, agriculture, and industrial processing. The assessment of the impairment of waterways for the various uses based on the ''indicator bacteria'' and Escherichia coli has not provided enough specificity in regard to health risk, sources of the pollution, identification of the responsible party, and control. This has fueled a demand for advanced pathogen detection.

In the twenty-first century, viral diseases have changed the landscape of medicine. Acquired immunodeficiency syndrome (AIDS) now infects millions of people worldwide and up to 30% of the populations in Africa. Waterborne diseases will be particularly devastating to these individuals and the list of potential waterborne viral agents is growing. Certain microbiological advances like the polymerase chain reaction (PCR) and microarray technology may provide the tools necessary for monitoring any new agent of interest. The era of pathogen discovery is not over. We should be prepared to use all the microbiological advances to continue to understand the transmission of diseases through water. This must then be coupled with our knowledge of engineering to determine how to maintain and develop the quantity and quality of our water resources. With the advances made in medicine, microbiology, and engineering we can add to the existing body of knowledge and continue to advance the field of ''environmental virology''.

The detection of viruses in water and other environmental samples constitutes special challenges. The standard method of detection of viral pathogens in environmental samples uses assays in mammalian cell culture. The infected cell cultures undergo observable morphological changes called cytopathogenic effects (CPEs) that are used for the detection of viruses. Even though many viruses are culturable in several cell lines and are thus detectable by the development of CPEs in cell culture, there are several viruses, like enteric waterborne adenoviruses types 40 and 41, which are difficult to culture and do not produce clear and consistent CPE. Other viruses, like waterborne caliciviruses, have not yet been successfully grown in cell cultures. Conventional cell culture assays for the detection of viruses in environmental samples have limited sensitivity and can be labor-intensive and timeconsuming. Two advances, the PCR and microarrays, have spurred the study of viruses and should be further applied to the field of environmental virology.

Since only a small percentage of viruses are cultivatable, there is a need to examine in a more systematic fashion the development of standard/consensus methods for the collection and processing of viruses in the water environment via molecular techniques. Table 1 lists some of the critical issues in the application of PCR techniques in environmental virology.

The PCR-detection assay allows for highly sensitive and highly specific detection of virus nucleic acid sequences. PCR was initially used as a research tool for the amplification of nucleic acid products. It is fast gaining acceptance in the clinical diagnostic setting and it has been effectively applied to the detection of viruses from environmental samples. The sensitivity of PCR has been demonstrated to be comparable or superior to cell culture (Raboni et al., 2003; Lee and Jeong, 2004) . In order to use PCR one must already know the exact sequence of a given region of the viral nucleic acid. Primers are then designed to amplify that specific region of the genome. Primers for the specific detection of many of the enteric viruses have been published. Nested and multiplex PCR, which are variations of the conventional PCR, have also been applied to virus detection in water samples. The chief drawback of PCR methods is that they are incapable of distinguishing between active and inactive targets.

The latest advancement in molecular methods is the development of quantitative real-time PCR. Quantitative PCR (qPCR) or real-time PCR can be used to quantify the original template concentration in the sample. Following DNA extraction, realtime PCR simultaneously amplifies, detects, and quantifies viral acid in a single tube within a short time. In addition to being quantitative, real-time PCR is also faster than conventional PCR. Real-time PCR requires the use of primers similar to those used in conventional PCR. It also requires oligonucleotide probes labeled with fluorescent dyes, or an alternative fluorescent detection chemistry different from conventional gel electrophoresis, and a thermocycler that can measure fluorescence. For quantification, generation of a standard curve is required from an absolute standard with known quantities of the target nucleic acid or organism. When realtime PCR quantitative results for adenoviruses in environmental samples were compared with conventional cell culture results, it was concluded that the real-time PCR method demonstrated higher quantities of adenoviruses in comparison with conventional techniques. This was likely due to the presence of noninfectious viruses and low sensitivities of tissue culture assays to serotypes present in the environmental samples (Choi et al., 2004; He and Jiang, 2004) .

Another modification of the standard PCR method, integrated cell culture (ICC)-PCR makes use of a cell culture step to enhance sensitivity and demonstrate infectivity. The ability to detect only infectious viruses among many noninfectious viruses is important for predictions of public health risk in water and other environmental samples, particularly after disinfection. As described above, molecular methods detect part of the viral DNA or RNA that indicates virus presence but not infectivity. To determine infectivity, molecular methods can be used in conjunction Table 1 Critical issues for PCR applications to environmental virology Sample concentration and inhibition control Environmental samples are less concentrated than clinical samples. Sample pre-concentration, usually by filtration and elution of large volumes of water, is required prior to DNA extraction. PCR in environmental samples is often inhibited by various substances. Sample dilutions and sample pretreatment methods should be evaluated for inhibition control.

For quantification, the use of real-time PCR is required along with generation of a standard curve from an absolute standard with known quantities of the target nucleic acid or organism. Real-time PCR requires the use of primers similar to those used in conventional PCR. It also requires oligonucleotide probes labeled with fluorescent dyes and a thermocycler that can measure fluorescence.

Instead of CPEs, detection of bacterial mRNA by reverse transcription (RT)-PCR assay can be used to detect infectivity. In the case of DNA viruses, detection of viral mRNA during cultivation is an indication of the presence of infectious viruses. In the case of RNA viruses detection of the doublestranded replicative form in cell cultures inoculated with viruses demonstrates infectivity . Specificity and primer and probe design PCR and real-time PCR primers should be designed to amplify only the DNA or RNA of interest. Realtime PCR primers differ from conventional PCR primers. The primer selection in the case of realtime PCR is restricted due to requirements of smaller target amplicon sizes than the PCR target sizes. Also, in the case of real-time PCR there is a need to select probes that meet certain criteria. QA/QC requirements The high sensitivity of the molecular techniques requires demanding quality assurance, quality control protocols. PCR techniques are very sensitive to contamination from amplified DNA. Contamination can result in false negatives and false positives. Appropriate lab infrastructure and highly trained personnel are required for effective QA/QC (EPA, 2004) . To avoid false-positive results, confirmation of amplification products, such as sequencing is required.

with cell cultures. The ability of viruses to infect cell cultures and to replicate their nucleic acid implies that they are also capable of infecting the human host. Targeting specific messenger RNA (mRNA), after inoculation of cell cultures with samples containing infectious viruses, cultures are observed for evidence of virus replication. Instead of CPEs, detection of viral mRNA by reverse transcription (RT)-PCR assay can sometimes be used to detect infectivity. DNA viruses, such as waterborne adenoviruses, form mRNA during replication in cells. Only infectious adenoviruses can enter cells and transcribe mRNA during replication; inactivated virus will be unable to do so. Therefore, detection of viral mRNA during cultivation is indicative of the presence of infectious viruses. Positive-strand RNA viruses, such as waterborne enteroviruses and noroviruses, produce a negative, complementary strand of RNA in the early phase of viral replication. This negative strand of RNA is bound to newly formed positive strands of RNA to form a double-stranded replicative form; detection of the replicative form in cell cultures inoculated with viruses can also be used to demonstrate infectivity .

Human adenoviruses are considered as emerging waterborne viruses. They are currently listed in the Environmental Protection Agency's Contaminate Candidate List.

The importance of adenoviruses and the potential health risks associated with their waterborne transmission has been recognized by the scientific community (Fong and Lipp, 2005) . Currently, there are 51 different types of human adenoviruses. Adenoviruses are a common cause of gastroenteritis, upper and lower respiratory system infections, and conjunctivitis (Swenson et al., 2003) . Other diseases associated with adenoviruses include acute and chronic appendicitis, cystitis, exanthematous disease, and nervous system diseases (Swenson et al., 2003) . Adenoviruses are considered important opportunistic pathogens in immunocompromised patients (Wadell, 1984) .

The potential of transmission of adenoviruses through water is suggested by the findings of several researchers. Enriquez et al. (1995) concluded that enteric adenoviruses are more stable in tap water and wastewater than poliovirus. Irving and Smith (1981) reported that adenoviruses are more likely to survive conventional sewage treatment than enteroviruses. In addition, Hurst et al. (1988) estimated that most adenoviruses detected in wastewater may be enteric adenoviruses. Borchardt et al. (2003) associated diarrhea of viral etiology (including adenoviruses) with drinking municipal water. Adenovirus outbreaks have also been associated with recreational exposure and swimming (Foy et al., 1968; Cardwell et al., 1974; D'Angelo et al., 1979; Martone et al., 1980; Turner et al., 1987; Papapetropoulou and Vantarakis, 1998; Harley et al., 2001) .

Adenoviruses have been found in wastewater samples in significant numbers in different geographic locations (Irving and Smith, 1981; Krikelis et al., 1985a, b; Hurst et al., 1988 , Puig et al., 1994 Greening et al., 2002; He and Jiang 2005) . Girones et al. (1995) presented data that showed the prevalence of adenoviruses in sewage samples. The presence of adenovirus has also been reported in river waters (Tani et al., 1995; Cho et al., 2000; Greening et al., 2002; Choi et al., 2004; Choi and Jiang, 2005; Haramoto et al., 2005; Van Heerden et al., 2005) and a river estuary (Castignolles et al., 1998) . Also, Jiang et al. (2001) detected human adenoviruses in urban runoff-impacted coastal waters in southern California, and Fong and Lipp (2004) detected adenoviruses in the coastal reaches of a river in Georgia.

Adenoviruses occurrence has been reported in drinking waters. Chapron et al. (2000) detected adenovirus in untreated surface waters collected and evaluated by the information collection rule. Fourteen of the 29 samples (48%) were positive for adenoviruses and 38% of these samples were determined to be infectious. Van Heerden et al. (2003) reported incidence of adenoviruses in raw and treated drinking water in South Africa. The results indicated human adenoviruses present in (13%) of the raw and (4%) of the treated water samples tested. At a later study, Van Heerden et al. (2005) reported adenovirus in 10 of 188 drinking water samples. Cho et al. (2000) and Lee and Kim (2002) detected adenoviruses in tap water.

To control the problem of adenovirus infections there is a need to further evaluate environmental exposure pathways and assess human exposure risk. Developing reliable and sensitive detection methods is crucial to this effort. It is also critical that the methods be able to quantify and differentiate between serotypes. Advances in molecular techniques have been applied in the detection of adenoviruses. Table 2 presents a summary of molecular techniques that have been applied to this complex group of viruses. Many of these assays have been used for detection in water. There is no approved, universally applied method for the detection of adenoviruses. Further methods development, comparison of existing techniques and evaluation of sensitivity and specificity of techniques is required to advance the state of knowledge relative to human adenoviruses and waterborne infections.

Microarrays were first described in 1995 by Schena et al. (1995) . They are arrays of spots on specially prepared glass or silicon surfaces. Each spot on an array serves as a single test at which a hybridization of DNA, immunological attachment, or chemical reaction can occur. These arrays can be useful for screening multiple samples against multiple targets but they are neither as sensitive nor as specific as PCR or cell culture. Microarray technology is enabled by the ability to deliver submicroliter volumes of material to an attachment surface or matrix. DNA microarrays are arrays in which DNA-DNA/DNA-cDNA or DNA-RNA hybridization reactions are an indication of a positive/negative reaction. Spotted DNA microarrays consist of either PCR generated or synthesized oligonucleotides that have been printed or mechanically spotted on to specially coated glass slides. In situ synthesized arrays have their probes chemically synthesized directly on to the support matrix. Yes PCR, Nested PCR Allard et al., 1990; Puig et al., 1994; Girones et al., 1995; Pina et al., 1998; Castignolles et al., 1998; Cho et al., 2000; Chapron et al., 2000; Greening et al., 2002; Maluquer de Motes et al., 2004; Rigotto et al., 2005; van Heerden et al., 2005; Choo and Kim, 2006 

Microarrays have conventionally been used to develop gene expression profiles of certain targets of interest. Increasingly, research has also focused on adapting microarray technology to screen clinical specimens against multiple target pathogens in a highly efficient manner (Zhou, 2003; Bodrossy and Sessitsch, 2004) . Microarrays have been designed for the detection and genotyping of hepatitis B virus, adenoviruses, Epstein-Barr virus, herpes simplex virus, influenza virus, and human papillomavirus (Sengupta et al., 2003; Boriskin et al., 2004; Korimbocus et al., 2005; Min et al., 2006; Song et al., 2006) .

Proposals have been put forth for using DNA microarrays as an environmental detection tool and possible biodefense tool (Pannucci et al., 2004; Sergeev et al., 2004) . Only a few examples exist for the application of microarray technology on environmental samples. For example, Kelly et al. (2005) have used DNA microarrays to analyze the nitrifying bacterial community in a wastewater treatment plant. Wu et al. (2004) developed a community genome array that was able to reveal species and strain differences in microbial community composition in soil, river, and marine sediments.

The use of microarrays as an environmental research tool can be divided into two broad categories: arrays that serve to detect specific gene sequences regardless of source and arrays that target specific pathogens. Straub and Chandler (2003) have proposed that a unified system for the detection of waterborne pathogens would significantly advance public health and microbiological water analysis and have indicated that advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. Table 3 summarizes some of the viral microarrays and their applications.

A number of commercially available microarray chip platforms are currently available. Their main differences are the manufacturing method employed and feature density. Affymetrix GeneChip arrays are able to accommodate up to 1.3 million unique features on a 5 00 Â 5 00 quartz wafer and are manufactured using a photolithographic masking technique (Pease et al., 1994) . Agilent microarrays have a 44,000 feature set and are synthesized using an inkjet printing method (Hughes et al., 2001) . Febit's Geniom microarrays contain only 6000 features but hybridization can be carried out with eight chips in parallel allowing multiple sample processing (Guimil et al., 2003) . Nimblegen microarrays contain 3,90,000 probes per array and are manufactured using a micromirror focusing technique. Less expensive glass slide arrays for smaller probe sets are also within the in-house fabrication capability of most research institutions (approximately 16,000 features per slide). Current limitations of the technology include issues regarding validation and low starting microbial biomass (Wu et al., 2006) and need to be addressed before the technology may be applied.

At present, the monitoring of public health occurs at the individual patient level. The highly disseminated nature of the public health system, however, means that it The method allowed us to discriminate OPV species from VZV, herpes simplex 1 virus (HSV-1), and HSV-2 that cause infections with clinical manifestations similar to OPV infections. Ryabinin et al. (2006) Respiratory pathogen

No data 20 common respiratory and 6 category A biothreat

The results demonstrate a novel, timely, and unbiased method for Wang et al. (2006) Able to detect the 3 major CNS disease-causing viruses from a single sample Korimbocus et al. (2005) Boriskin et al. (2004) takes either a long time or a massive influx of cases before a disease outbreak is recognized. The trend towards increasingly urbanized and dense city living and the more frequent travel between communities necessitates that community health monitoring adopts a more proactive preventative role instead of merely recording and reporting disease data. In order to do so, there need to be tools that are able to screen for the large panel of possible viral pathogens which are representative of the pathogen loads present in the larger community. To meet this requirement it becomes logical to monitor the community's sewage using microarrays designed to detect the presence of waterborne pathogens. Using the OligoArray version 2.1 software written by Rouillard et al. (2003) , Wong et al. (2006) have designed a total of 780 probes to detect 25 of the common virus families known to cause gastroenteritis. Two additional probes were placed unto the microarray as quality control sequences for fabrication. The 25 virus families targeted in this micoarray are hepatitis A and E virus, human adenovirus A-F, noroviruses, sapoviruses, human enterovirus groups A-E, polioviruses, rotavirus groups A-C, coronaviruses, astroviruses, human cytomegalovirus, torovirus, polyomaviruses, and picobirnaviruses. These oligonucleotide probes were synthesized unto a 63 Â 116 microarray by the Gulari Research Group at the University of Michigan (Gao et al., 2001) . Figure 1 illustrates the processing steps required to prepare a sample for hybridization on an array. Environmental samples are first concentrated using conventional virus concentration methods like organic flocculation, tangential flow filtration, or ultrafiltration. Concentrates are then biologically amplified by passage through cell culture and virus nucleic acid is extracted. Labeling of viral nucleic acid is Fig. 1 The processing steps required to prepare a sample for hybridization on an array. carried out through the use of DNA polymerase or RT enzymes. The labeled nucleic acid can then be hybridized on the microarray and a distinctive hybridization pattern is produced when target viruses bind to their complementary probes on the array.

Testing of the virus microarray was carried out using poliovirus virus LSC-1 and adenovirus type 40 and 41 as test subjects. Poliovirus was cultured on Buffalo green monkey (BGM) cells and adenoviruses were cultured on MA104 cells and their respective nucleic acid contents were extracted. Poliovirus RNA was labeled using the Superscript Indirect Labelling System (Invitrogen, Inc. Carlsbad, CA). Adenovirus DNA was labeled using the large fragment of DNA polymerase I (Klenow Fragment). Hybridization was carried out using an in-house hybridization wash station at 201C for approximately 17 h. Subsequently, the microarray was scanned using a Molecular Devices model 4000B Genepix Scanner (Molecular Devices Corporation, Sunnyvale, CA), washed at 251C, scanned, and subsequently washed and rescanned at 11C intervals to generate a washing curve profile. Figure 2 illustrates the hybridization profile obtained from the hybridization of poliovirus, adenovirus type 40 and 41 with the virus microarray. Excellent signal to noise was achieved and the viruses were easily identified and distinguished.

Current work demonstrates that the chip can be used with viral concentrates from cell culture systems originating from sewage, with unique virus detection in community wastewater indicative of infections in the population. Thus future applications suggest, as with PCR, it will be a combination of conventional methods used in environmental virology and new techniques that will enhance pathogen discovery.

Viruses remain a public health concern and should remain a priority for the water and health community. These bio-nano particles are excreted in high concentrations by infected individuals, have high potency (probability of infection is high with low numbers (Haas et al., 1999) ), and are environmentally robust. The ability of both DNA viruses and RNA viruses to rapidly evolve means new and emerging viral pathogens will need to be addressed. Pathogen discovery and characterization, occurrence in the environment, exposure pathways, and health outcomes via environmental exposure need to be addressed. This will likely follow a new microbial risk framework, which will require focused research on some important properties of viral disease transmission. The future will require models that examine community risks and provide explicit links between the models currently under development for environmental exposure and infectious disease. It is predicted that there will be in each of these areas some key scientific issues that will need to be addressed (Table 4 ). New advances like qPCR and microarray technology should be applied to wastewater streams for viral pathogen discovery and for characterization of disease in our populations. These data should be used to further an analysis of water quality and health status. Adenoviruses, polyomaviruses, noroviruses, and other respiratory viruses need to be characterized, as does respiratory viral transmission particular via recreational exposure. Greater assessment of the occurrence of animal viruses in water, their role in animal health, and application to source tracking are needed. Finally, environmental virology needs to include the detection, occurrence, and transmission of viruses from fomites. The health impacts of these viral pathogens in nontraditional outcomes such as cancer are future areas requiring research.

Polymerase chain reaction for detection of adenoviruses in stool samples

Microarray-based detection and typing of foot-and-mouth disease virus

Oligonucleotide microarrays in microbial diagnostics

Septic system density and infectious diarrhea in a defined population of children

DNA microarrays for virus detection in cases of central nervous system infection

Epidemic with adenovirus type 7 acute conjuctivitis in swimmers

Detection of adenovirus in the waters of the Seine River estuary by nested-PCR

Detection of astroviruses, enteroviruses, and adenovirus types 40 and 41 in surface waters collected and evaluated by the information collection rule and an integrated cell culture-nested PCR procedure

Combining multiplex reverse transcription-PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16

Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult

Detection of adenoviruses and enteroviruses in tap water and river water by reverse transcription multiplex PCR

Real-time PCR quantification of human adenoviruses in urban rivers indicates genome prevalence but low infectivity

Application of Real-Time PCR and Tissue Culture Assay for Adenovirus Detection in Two Southern California Urban Rivers

Detection of human adenoviruses and enteroviruses in Korean oysters using cell culture, intergrated cell culture-PCR, and direct PCR

Design of microarray probes for virus identification and detection of emerging viruses at the genus level

Development of Molecular Methods to Detect Infectious Viruses in Water

Pharygoconjunctival fever caused by adenovirus type 4: report of a swimming pool-related outbreak with recovery of virus from pool water

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses

Survival of the enteric adenoviruses 40 and 41 in tap, sea, and waste water

Quality assurance/quality control guidance for laboratories performing PCR analyses on environmental samples. US EPA, Office of Water

Molecular Detection of Waterborne Enteric Viruses in the Coastal Reaches of the Altamaha River

Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom

Adenovirus type 3 epidemic associated with intermittent chlorination of a swimming pool

A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids

Viruses in source and drinking water

Detection of adenovirus and enterovirus by PCR amplification in polluted waters

Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples

Multiplexed, real-time PCR for quantitative detection of human adenovirus

Geniom technology-the benchtop array facility

Quantitative Microbial Risk Assessment

Application of cation-coated filter method to detection of noroviruses, enteroviruses, adenoviruses, and torque teno viruses in the Tamagawa River in Japan

A primary school outbreak of pharyngoconjunctival fever caused by adenovirus type 3

Quantification of human adenoviruses and enterococcus in environmental water samples

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pring-Akerblom P. Rapid and quantitative detection of human adenovirus DNA by real-time PCR

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer

Comparison of cytopathogenicity, immunofluerescence and in situ DNA hybridization as methods for the detection of adenoviruses

One-year survey of enteroviruses, adenoviruses, and reoviruses isolated from effluent at an activated-sludge purification plant

Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California

Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

Quantitative realtime PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41

DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples

Transcriptomal analysis of varicella-zoster virus infection using long oligonucleotide-based microarrays

Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR

Rapid detection of infectious adenoviruses by mRNA real-time RT-PCR

DNA probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses

Detection of indigenous enteric viruses and raw sewage effluents of the City of Athens, Greece, during a two-year survey

Seasonal distribution of enteroviruses and adenoviruses in domestic sewage

Use of cell culture-PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a tool to monitor infectious adenoviruses and enteroviruses in river water

Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliability of waterborne virus detection

Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea

Detection of Epstein-Barr virus infection and gene expression in human tumors by microarray analysis

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses

Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays

Validation of a fully integrated microfluidic array device for influenza a subtype identification and sequencing

Real-time reverse transcription-PCR for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children

A universal microarray for detection of SARS coronavirus

Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination

An outbreak of adenovirus type 3 disease at a private recreation center swimming pool

Oligonucleotide microarray with RD-PCR labeling technique for detection and typing of human papillomavirus

A large waterborne viral hepatitis E epidemic in Kanpur

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

Virulence signatures: microarray-based approaches to discovery and analysis

Detection of adenovirus outbreak at a municipal swimming pool by nested PCR amplification

Global illness and deaths caused by rotavirus disease in children

Prevalence of vaccinederived polioviruses in sewage and river water in South Africa

Light-generated oligonucleotide arrays for rapid DNA sequence analysis

Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay

Viral Pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification

Human-blind probes and primers for dengue virus identification

Comparison of PCR, enzyme immunoassay and conventional culture for adenovirus detection in bone marrow transplant patients with hemorrhagic cystitis

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR)

A simple and rapid single-step multiplex RT-PCR to detect norovirus, astrovirus and adenovirus in clinical stool samples

OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach

Microarray assay for detection and discrimination of orthopoxvirus species

Comprehensive detection and serotyping of human adenoviruses by PCR and sequencing

Quantitative monitoring of gene expression patterns with a complementary DNA microarray

Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization

Multipathogen oligonucleotide microarray for environmental and biodefense applications

New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotidemicroarray

Genotyping of hepatitis B virus (HBV) by oligonucleotides microarray

Protecting public health from the impact of body-contact recreation

Towards a unified system for detecting waterborne pathogens

Manual of Clinical Microbiology

Seasonal distribution of adenoviruses, enteroviruses and reoviruses in urban river water

Experimental evaluation of the FluChip diagnostic microarray for influenza virus surveillance

Community outbreak of adenovirus type 7a infections associated with a swimming pool

United Nations Educational, Scientific and Cultural Organizations

Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant

Hearing on Meeting the Nation's Wastewater Infrastructure Needs

Incidence of adenoviruses in raw and treated water

Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa

Environmental Protection Agency water quality guidelines for recreational waters prevent gastrointestinal illness? A systematic review and meta-analysis

Molecular epidemiology of human adenoviruses

Identifying influenza viruses with resequencing microarrays

Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay

Development of a Virulence Factor Biochip and its Validation for Microbial Risk Assessment in Drinking Water

Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications

Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification

Species-specific identification of human adenoviruses by a multiplex PCR assay

Microarrays for the detection of HBV and HDV

Microarrays for bacterial detection and microbial community analysis