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Research and Development - Drinking Water

Title: Detection of Enteric Viruses in Archived ICR Sample Concentrates Using an Integrated Cell Culture-Nested PCR Technique.

Authors: Paul S. Warden, Nicola A. Ballester, Amy E. Moore, Justin H. Fontaine, Kristen S. Fallon and Aaron B. Margolin

Abstract:
The preamble of the Information Collection Rule (ICR) referenced the goal of archiving virus samples for future study. From samples submitted to our laboratory during the ICR, Analytical Services, Inc. (ASI) and the University of New Hampshire (UNH) created a repository of sample concentrates that were representative of a broad geographic area and were consistent with respect to methods of sample collection, handling and storage. The objective of this study was to examine viral occurrence in archived source water samples using a sophisticated, sensitive molecular technique and to compare these results to the respective data obtained from the Total Cultivatable Virus Assay (TCVA) method used in the ICR.

Ninety-one of these sample concentrates were subsequently analyzed using Integrated Cell Culture/nested Polymerase Chain Reaction (ICC/nPCR) for the detection of adenovirus 40 and 41, astrovirus, and enteroviruses (Coxsackie virus, echovirus and poliovirus). Samples were selected for inclusion in the study to maximize geographic distribution and to include approximately 50% TCVA-positive and 50% TCVA-negative samples. The analytical procedures employed for ICC/nPCR were as previously published (Chapron, et. al, 2000). Infectivity was determined for all ICC/nPCR positive samples. All positive results were confirmed by re-analysis. A Performance Evaluation (PE) program was implemented with the USEPA, primarily to assess the potential for false-positive results.

Of the 91 samples selected for inclusion in the study, 41 were positive by the TCVA method. In contrast, 50 of 91 samples (an increase of 22%) were positive for one or more viral types using ICC/nPCR. Of these 50 ICC/nPCR positive samples, 34 were confirmed to contain one or more types of infectious viruses. Significantly, over half of the TCVA-negative samples (29/50 or 58.0%) were positive for one or more viral types by ICC/nPCR. Sixteen of these samples (16/50 or 32.0%) were confirmed by ICC/nPCR to contained infectious viruses. Interestingly, of the 41 TCVA-positive samples, only 21 (51.2%) were positive by ICC/nPCR; the other 20 were negative. This could be caused by one or more of the following reasons: age of the sample (samples had been stored at -80o C for 2-4 years); primer selection (reovirus primers were not included, yet reoviruses are supported on BGMK cells); and the presence of other non-human viruses known to cause CPE on BGMK cells (not detected by the primers used in our assay).

Even without reovirus included in this study, the detection of infectious enteric viruses in over half of the samples presumed negative by traditional cell culture methods challenges the current assumptions regarding viral occurrence in source waters of public water supplies in the United States and the public health risk assessments based on these assumptions. Adenoviruses, Coxsackie virus and echovirus are included in the USEPA Contaminant Candidate List (CCL) because of evidence of attributable outbreaks, the need for method development and/or the need for occurrence data. Further research is needed using ICC/nPCR, or equally sensitive techniques, to determine the extent of viral occurrence in source and finished waters.

Objectives

• To determine the occurrence of selected human enteric viruses in archived source water sample concentrates.

• To compare the ICR (TCVA) and ICC/nPCR data to evaluate these methods as measures of the occurrence of the enteric viruses.

Methods
Samples were screened for adenovirus, astrovirus, rotavirus, and enteroviruses by ICC/nPCR using BGMK and Caco-2 cell lines and specific primers for each viral group.

The infectious status of samples that were positive by ICC/nPCR was determined by analyzing sample concentrates by direct PCR (or RT-PCR) and comparing these results to ICC/nPCR.

If the ICC/nPCR reaction was positive but the PCR reaction on the concentrate was negative, the sample was suspected to contain infectious viruses of the given type. The laboratory procedures that were followed have been previously published (Chapron, et. al., 2000) and are described in the WQTC proceedings, including QA/QC, the study Performance Evaluation program, and interference/inhibition trials.

Discussion
Viral occurrence - More samples were designated as “positive” using ICC/nPCR than TCVA, which may be due to increased sensitivity of ICC/nPCR and the detection of non-infectious virus particles and/or the presence of adenovirus, astrovirus and/or rotavirus, which do not form CPE on BGMK cells.

Figure 1. Percent of total samples (n = 91) positive and infectious by ICC/nPCR (TCVA for reference).

However, 49% of the TCVA positive samples were negative by ICC/nPCR. Factors that may have contributed include:

	Sample Age - The samples were between two and four years old at the time of this study. The titer of viral stocks are known to decrease over time, and it is reasonable to suspect that some degradation may have occurred in the sample concentrates.
	Non-target viruses – Some TCVA positives may be due to reoviruses, which cause CPE on BGMK cells. It is suspected that there are also non-human animal viruses that are supported on BGMK cells and may cause CPE.
	Distribution of Viral Particles – Non-uniform distribution of viral particles in samples concentrates may have confounded the data.

Figure 2. Percent of TCVA positive samples (n = 41) determined to be positive and infectious by ICC/nPCR (TCVA for reference).

However, enteroviruses were the most common viral type detected by ICC/nPCR in the TCVA negative samples. All of these positive results were confirmed to eliminate the possibility of false-positives. Therefore, we believe these enterovirus results indicate the sensitivity of the molecular assay compared to the traditional cell culture assay.

Figure 3. Percent of TCVA negative samples (n = 50) determined to be positive and infectious by ICC/nPCR.

No study samples were positive for rotavirus by ICC/nPCR, although routine samples and controls were normal. This may indicate the need for optimizing the method for archived samples, rather than rotavirus absence, and may have caused the overall virus occurrence rate to have been underestimated.

Viral Infectivity
Infectious viruses were detected in fewer samples by ICC/nPCR than TCVA (34 vs. 41 of 91, respectively). The three factors discussed above may have contributed to these results.

Viruses were detected by ICC/nPCR in 58% of the 50 TCVA negative samples (32% infectious). If this statistic is validated in a more comprehensive survey, it would be a very significant finding that over half of the sources waters are contaminated with viruses and approximately one-third contain infectious viruses.
Enteroviruses were the most commonly detected infectious virus by ICC/nPCR (25 samples), followed by adenovirus (17) and astrovirus (6). However, when examined as a ratio of infectious to positive (by ICC/nPCR); adenovirus has the highest frequency (17 infectious of 24 positive or 70.8%), then enterovirus (25 of 38 or 65.8%) and then astrovirus (6 of 18, or 33.3%). These data may suggest differential viral infectivity rates in the environment and lend increased importance to the study of adenoviruses.

The rates of viral occurrence, and the rate of occurrence of infectious viruses indicated in this study are substantial and are higher than the rates reported from the ICR. Even without reovirus included in this study, the detection of infectious enteric viruses in over half of the samples presumed negative by traditional cell culture methods challenges the current assumptions regarding viral occurrence in sources waters of the public water supplies in the United States and the public health risk assessments based on these assumptions.

Acknowledgements
This study was funded by the U.S. EPA. (Order No. 0C-W025-NASA). The authors are grateful to Mary Ann Feige and Dr. Shay Fout, who provided project guidance.

References
USEPA. 1998. Announcement of the Drinking Water Contaminant Candidate List. Federal Register. 63:10274-10287.

USEPA. 1999. Revisions to the Unregulated Contaminant Monitoring Regulation for Public Water Systems; Final Rule. Federal Register. 64: 50555-50620.

Chapron, C.D., N.A. Ballester, J.H. Fontaine, C.N. Frades and A.B. Margolin. 2000. The Detection of Astrovirus, Enteroviruses and Adenovirus Type 40 and 41 in Surface Waters Collected and Evaluated by the Information Collection Rule and Integrated Cell Culture/Nested PCR Procedure. Applied and Environmental Microbiology, Vol. 66, no. 6, .2520-2525.

Gerba, C. 2001, personal communication (to A. Margolin).

Pina, S. M. Puig, F. Lucena, J. Jofre and R. Girones. 1998. Viral Pollution in the Environment and in Shellfish: Human Adenovirus Detection by PCR as an Indicator of Human Viruses. Applied and Environmental Microbiology, Vol. 64, No. 9, p. 3376-3382.

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