Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus

• Published in: Journal of virological methods Vol. 219; no. C; pp. 51 - 61
• Main Authors: Karim, Mohammad R., Fout, G. Shay, Johnson, Clifford H., White, Karen M., Parshionikar, Sandhya U.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2015; Elsevier
• Subjects: Animals; Azides - pharmacology; Caliciviridae Infections - diagnosis; Caliciviridae Infections - virology; Enteric virus; Enterovirus; Enterovirus - drug effects; Enterovirus - genetics; Enterovirus Infections - diagnosis; Enterovirus Infections - virology; Humans; Norovirus; Norovirus - drug effects; Norovirus - genetics; Norwalk virus; Poliovirus; Propidium - analogs & derivatives; Propidium - pharmacology; Propidium monoazide; Real-Time Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - genetics; Viral Plaque Assay; Virus Inactivation - drug effects; Virus Inactivation - radiation effects; UV; Propidium monoazide; Norovirus; Enteric virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2015.02.020

•PMA RT-PCR differentiates between infectious and heat and chlorine inactivated poliovirus.•PMA RT-qPCR prevents amplification of chlorine inactivated MNV, but not Norwalk virus.•PMA RT-PCR is not effective for UV inactivated viruses.•PMA assays prevent amplification of MNV MNV-1 RNA; thus positive results come from intact virions. 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.