Integrated cell culture/PCR for detection of enteric viruses in environmental samples

Abstract

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens. CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone. Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.