Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

• Published in: Applied and Environmental Microbiology Vol. 76; no. 13; pp. 4318 - 4326
• Main Authors: Parshionikar, Sandhya, Laseke, Ian, Fout, G. Shay
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.2010; American Society for Microbiology (ASM)
• Subjects: Azides - pharmacology; Biological and medical sciences; Coxsackievirus; Deoxyribonucleic acid; DNA; Drinking water; Dyes; Echovirus; Enterovirus B, Human - genetics; Enterovirus B, Human - isolation & purification; Enterovirus B, Human - pathogenicity; Fundamental and applied biological sciences. Psychology; Genes; Hot Temperature; Humans; Hypochlorous Acid - pharmacology; Methods; Microbiology; Norwalk virus; Norwalk virus - genetics; Norwalk virus - isolation & purification; Norwalk virus - pathogenicity; Poliovirus - genetics; Poliovirus - isolation & purification; Poliovirus - pathogenicity; Propidium - analogs & derivatives; Propidium - pharmacology; Reverse Transcriptase Polymerase Chain Reaction - methods; Ribonucleic acid; Rivers - virology; RNA; RNA Viruses - drug effects; RNA Viruses - genetics; RNA Viruses - isolation & purification; RNA Viruses - pathogenicity; RNA, Viral - analysis; RNA, Viral - isolation & purification; Virology; Viruses; Water; Infection; Polymerase chain reaction; Virus; Nucleotidyltransferases; RNA-directed DNA polymerase; Enzyme; Transferases; Sample
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02800-09

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.