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Viral Analyses

Technical Document

Introduction

Outbreaks of foodborne and waterborne illness have been associated with viruses^1,2,3. The viruses that are typically transmitted by water (via the fecal-oral route) belong to one of the largest groups of viruses called the enteric viruses. At least 140 human enteric virus types are known, including 72 serotypes of enteroviruses, adenoviruses, reoviruses, rotaviruses, norwalk virus, and others². Most of the enteric viruses are responsible for causing gastroenteritis or hepatitis, but some can affect the central nervous system, heart muscle, and respiratory system.

Analytical Services, Inc. (ASI)/University of New Hampshire (UNH) was one of ten microbiology laboratory entities approved by the United States Environmental Protection Agency (EPA) to participate in virus sample analysis for the Information Collection Rule (ICR). ASI’s Senior Scientist, Dr. Aaron Margolin, Associate Professor at UNH, was awarded the EPA contract to develop and test the protocol for preparation of the ICR virus Performance Evaluation (PE) samples.

Viral Sampling

Viral sampling is accomplished by following Standard Method #9510C (Virus Concentration from Large Sample Volumes by Adsorption to and Elution from Microporous Filters) from Standard Methods for the Examination of Water and Wastewater⁴. This procedure involves pumping large volumes of water through a cartridge filter to concentrate viral particles and elution to desorb viral particles from the filter. Detection of viruses may be accomplished using cell culture, polymerase chain reaction (PCR), or other detection methods.

Cell Culture

Viruses which are pathogenic to humans have traditionally been detected by cell culture methods. These methods involve culturing host cells as stationary monolayers in sterile petri dishes and infecting the cells with a viral inoculum. Viruses are visualized microscopically by morphological changes in the infected cells (cytopathogenic effects). Cell culture media components are expensive, the technique is relatively difficult to perform, and results are often not available for up to 6 weeks after commencing the assay. In addition, viruses are host specific and require different cell lines for successful infection and replication. ASI has maintained its reputation as an exceptional virology cell culture laboratory since 1990.

Polymerase Chain Reaction (PCR)

Another method of detection ASI offers is polymerase chain reaction (PCR). PCR is a molecular method that amplifies viral nucleic acid without the need of culture techniques^{5, 6}. ASI routinely analyzes drinking water and biosolids samples for enteroviruses (Poliovirus, Coxsackie virus, and Echovirus types), Hepatitis A virus, Rotavirus, and Norwalk virus using PCR. PCR analysis for enteroviruses involves the use of "panentero" primers, which generate one result (positive or negative) for the entire group⁵. At this time, genus specific PCR for non-Poliovirus enteroviruses is not routinely available. PCR does not indicate whether or not the viral particles are infectious, but does indicate the presence or absence of viral nucleic acid. There are several advantages to using PCR including its sensitivity of viral detection, rapid generation of results, and cost effectiveness. These qualities support its use as a screening tool.

Currently, ASI is the only laboratory in the United States licensed by Perkin Elmer Corporation and Roche Molecular systems, Inc. to perform commercial PCR analyses on environmental samples. Under this licensing agreement, in addition to performing PCR-based research, we are able to report site specific results upon which decisions can be made.

Integrated Cell Culture /PCR

Recent methodological advances have focused on an integrated cell-culture/PCR (ICC/PCR) assay which allows for the detection of infectious virus. The method involves inoculation of cell monolayers with virus, incubation of the culture flasks for 5-7 days, and performing RT-PCR on the cell lysate and supernate. Compared to standard PCR, one significant advantage of this assay is that it allows for the differentiation between infectious and noninfectious viruses by detecting only viruses which have successfully infected and replicated in the cell monolayers. Compared to standard cell culture, there are several other advantages including rapid turnaround time (8-10 days), reduced labor, reduced cost, as well as detection of only infectious viruses. We are currently validating our CC/PCR methodology for the detection of enteroviruses (Poliovirus, Coxsackie virus and Echovirus types), Hepatitis A virus, and Rotavirus in water.

Bacteriophages

Bacteriophages are viruses that infect bacterial cells. Phages have served as useful models for the behavior of human enteric viruses in water treatment processes because of their similarity to enteric viruses in structure, size, and resistance to inactivation¹. In addition, phages are inexpensive to propagate in high quantities, detection methods are easy, and results are available within 24 hours. EPA is currently evaluating whether or not to include source water monitoring for bacteriophages (as viral indicators) for the upcoming Ground Water Rule. ASI provides bacteriophage field sampling and analysis for clients who wish to monitor their source waters for bacteriophages.

Given the "user-friendly" nature and cost effectiveness of assaying bacteriophages as compared to animal viruses, drinking water treatment unit (DWTU) testing studies performed at ASI frequently employ bacteriophage MS2 (which infects Escherichia coli) as the challenge organism. Bacteriophage 41C, a phage that infects Bacillus subtilis, has also been used in challenge studies at both the bench and pilot scale levels.

References

1. Hedberg, C.W. and M.T. Osterholm. 1993. Outbreaks of FoodBorne and Waterborne Viral Gastroenteritis. Clinical Microbiology Reviews, 6:199-210.

2. Hurst, C.J. 1991. Presence of Enteric Viruses in Freshwater and Their Removal by the Conventional Drinking Water Treatment Process. WHO Bulletin OMS, 69:113-119.

3. LeBaron, C.W. et al. 1990. Viral Agents of Gastroenteritis: Public Health Importance and Outbreak Management. MMWR, 39:1-24.

4. APHA, AWWA, WEF. Standard Methods for the Examination of Water and Wastewater. (20^th ed. 1998).

5. Abbaszadegan, M. et al. Detection of Enteroviruses in Groundwater with the Polymerase Chain Reaction. AEM, 59:1318-1324.

6. Regan, P.M and A.B. Margolin. 1997. Development of a Nucleic Acid Capture Probe with Reverse Transcriptase-Polymerase Chain Reaction to Detect Poliovirus in Groundwater. J. Virol. Mthds, 64:65-72.

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