Overview

Enteric viruses have been detected in environmental waters and potable water supplies. Water supply safety is evaluated by examination for bacteria that are not accurate surrogates for viruses that pose a public health threat in low levels. Current viral detection methods include standard techniques for in vitro cultivation in cell cultures and newer molecular methods for viral nucleic acid detection. The new methods are rapid and sensitive, but detect only the viral nucleic acid and do not determine infectivity. Methods for the detection of potentially infectious viruses are needed. The objectives of this project were to: develop and evaluate sensitive, selective methods for infectious virus capture from water concentrates; develop and evaluate cell culture combined with rapid, sensitive RT-PCR and PCR methods for detection of infectious viruses; develop methods for rapid, sensitive, and specific quantitation of viruses; validate the performance of methods for water sample concentrates containing assay inhibitory substances; validate the ability of methods to distinguish infectious from non-infectious viruses inactivated by disinfectants with different inactivation mechanisms, free chlorine, and low pressure UV radiation; and collect, concentrate, and archive raw and finished water samples for subsequent virus analysis New molecular tests such as RT-PCR or PCR may yield ambiguous positive results by detecting noninfectious or inactivated viruses. To overcome this problem, this study employed additional processing steps that depend on functional components of the virus needed for infection as an approach to detecting only infectious viruses. These methods were intended to preclude the detection of noninfectious viruses and increase the potential for detecting infectious viruses. Methods included the specific capture of virus by cellular receptors for virus in vitro, followed by molecular detection of viral nucleic acid in the captured virus. Another method demonstrated was the selection and detection of specific nucleic acid only present in the cell during virus replication. In addition, rapid real-time molecular tests that may be integrated with methods for the functional components were developed. The following are highlights from the project: Methods for detection of ribonucleic acid forms only present in inoculated cells after viral infection were developed and validated for two different types of viruses potentially contaminating water sources - enteroviruses and Adenoviruses. A purified cellular protein necessary for attachment of Coxsackie B viruses to cells was used to capture potentially infectious viruses for detection by RT-PCR. Real-time RT-PCR and PCR assays, complete in 30-90 minutes, were developed for enterovirus, hepatitis A virus, Adenovirus, and Norovirus detection. These assays can be used in conjunction with some of the other developed methods to detect infectious virus. The increasing need for the reuse of water and the inadvertent reuse of natural sources of water impacted by point and non-point human waste discharges make detection of infectious viruses in source and finished water supplies an important need. The researchers have developed and demonstrated several new methods for the selective detection of potentially infectious, pathogenic viruses transmitted by water. Rapid real-time molecular detection methods were developed that can be combined with the infectivity assays for rapid detection of infectious viruses. Further work can expand these methods to other viruses. These assays can be used in the future at utilities when new technology is commercially available for discrimination of infectious and non-infectious viruses.

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