Evaluation of molecular methods for the discrimination of infectious enteric viruses in complex water matrices

Human pathogenic enteric viruses in surface water pose a significant threat to the public health since a complete viral removal cannot be guaranteed with state of the art waste water treatment plants. Evaluation of the viral load – especially in regards of infectious viruses - of a water body is particularly difficult since molecular biological techniques like quantitative polymerase chain reaction (qPCR) do not allow the distinction between infectious and non-infectious viruses. Animal cell culture – the golden standard method of virus detection – allows this distinction but commercial unavailability of cell lines for relevant viruses (norovirus) in addition to high time and material consumption limits it significantly.

In the past years, several innovative approaches to develop alternative methods of viral detection in environmental water have been pursued by the department of hygiene, social- and environmental medicine of the Ruhr-University Bochum. The application of cell membrane impermeable intercalating dyes Ethidium and Propidium Monoazide (viability or capsid integrity qPCR), which inhibit the amplification of viral particles with compromised capsid has been examined both in lab scale as in complex water matrices (Leifels et al., 2015, Leifels et. al., 2016) as well as the usage of the xMAP Luminex flow cytometry (Hamza et al., 2014). Both new methods show advantages in comparison to qPCR but are not solely capable of replacing it.

The aim of this dissertation is to further evaluate the application of novel methods of virus detection in surface water with simultaneous distinction of viral infectivity by increasing the sensitivity and applicability of the capsid integrity qPCR as well as the utilization of a long amplicon or comparative qPCR, which aims at regions of the viral genome with different persistence towards disinfection techniques like the exposure to UV light or the addition of chlorine.