Capsid Integrity qPCR—An Azo-Dye Based and Culture-Independent Approach to Estimate Adenovirus Infectivity after Disinfection and in the Aquatic Environment

Mats Leifels,Angela Hagemeier,Nicholas J Ashbolt,Emanuele Sozzi,David Shoults,Lars Jurzik,Alyssa Wiedemeyer

doi:10.3390/w11061196

Abstract

Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings.

Highlights

Wastewater treatment technology has markedly improved in efficacy over the past decades [1].Waterborne diseases caused by human or animal fecal contamination, remain a threatWater 2019, 11, 1196; doi:10.3390/w11061196 www.mdpi.com/journal/waterWater 2019, 11, 1196 to public health worldwide [2,3,4]
human adenoviruses (HAdV)-Hernroth amplicons are only half the length of HAdV-Heim, the ability of propidium monoazide (PMA) and ethidium monoazide (EMA) pretreatment to remove non-infectious virions (“false-positive” results) was observed to be similar
As for bacteria, similar observations have been described for a variety of enteric viruses and bacteriophages after exposure to UV light by [38,59,60,61]

Summary

Introduction

Wastewater treatment technology has markedly improved in efficacy over the past decades [1].Waterborne diseases caused by human or animal fecal contamination, remain a threatWater 2019, 11, 1196; doi:10.3390/w11061196 www.mdpi.com/journal/waterWater 2019, 11, 1196 to public health worldwide [2,3,4]. Waterborne diseases caused by human or animal fecal contamination, remain a threat. The quality of surface water used for recreational activities and drinking water production relies on fecal bacterial indicators such as Escherichia coli and enterococci [5,6]. Human enteric viruses tend to drive infection risk for exposures to waterborne pathogens [11,12,13,14,15]. There is an increasing focus from government regulators and scientists to identify useful viral indicators [16,17,18]. While nearly 150 waterborne enteric viruses have been associated with human illnesses [19], human adenoviruses (HAdV) are considered to be one of the most appropriate indicators for human fecal viral presence in waters [7]

Methods

Results

Conclusion