Critical issues in application of molecular methods to environmental virology.

Waterborne diseases have significant public health and socioeconomic implications worldwide. Many viral pathogens are commonly associated with water-related diseases, namely enteric viruses. Also, novel recently discovered human-associated viruses have been shown to be a causative agent of gastroenteritis or other clinical symptoms. A wide range of analytical methods is available for virus detection in environmental water samples. Viral isolation is historically carried out via propagation on permissive cell lines; however, some enteric viruses are difficult or not able to propagate on existing cell lines. Real-time polymerase chain reaction (qPCR) screening of viral nucleic acid is routinely used to investigate virus contamination in water due to the high sensitivity and specificity. Additionally, the introduction of metagenomic approaches into environmental virology has facilitated the discovery of viruses that cannot be grown in cell culture. This review (i) highlights the applications of molecular techniques in environmental virology such as PCR and its modifications to overcome the critical issues associated with the inability to discriminate between infectious viruses and nonviable viruses, (ii) outlines the strengths and weaknesses of Nucleic Acid Sequence Based Amplification (NASBA) and microarray, (iii) discusses the role of digital PCR as an emerging water quality monitoring assay and its advantages over qPCR, (iv) addresses the viral metagenomics in terms of detecting emerging viral pathogens and diversity in aquatic environment. Indeed, there are many challenges for selecting methods to detect classic and emerging viruses in environmental samples. While the existing techniques have revealed the importance and diversity of viruses in the water environment, further developments are necessary to enable more rapid and accurate methodologies for viral water quality monitoring and regulation.