title: Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.
abstract: Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.
authors: Technical Support Center, Office of Ground Water and Drinking Water, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
authors: Mohammad R
authors: MR
authors: Karim
authors: Biohazard Assessment Research Branch, Microbiological and Chemical Exposure Assessment Research Division, National Exposure Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA. Electronic address: fout.shay@epa.gov.
authors: G Shay
authors: GS
authors: Fout
authors: Microbial Contaminant Control Branch, Water Supply and Water Resources Division, National Risk Management Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
authors: Clifford H
authors: CH
authors: Johnson
authors: Microbial Contaminant Control Branch, Water Supply and Water Resources Division, National Risk Management Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
authors: Karen M
authors: KM
authors: White
authors: Technical Support Center, Office of Ground Water and Drinking Water, United States Environmental Protection Agency, 26 W, Martin Luther King Dr., Cincinnati, OH 45268, USA.
authors: Sandhya U
authors: SU
authors: Parshionikar
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology*; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology; D004770:Enterovirus / Q000187:drug effects* / Q000235:genetics*; D004769:Enterovirus Infections / Q000175:diagnosis / Q000821:virology; D006801:Humans; D029322:Norovirus / Q000187:drug effects* / Q000235:genetics*; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D010948:Viral Plaque Assay; D038301:Virus Inactivation / Q000187:drug effects / Q000528:radiation effects
chemical_list: D001386:Azides; D012367:RNA, Viral; C533957:propidium monoazide; D011419:Propidium
keywords: Enteric virus; Norovirus; Propidium monoazide; UV