Enhancing Quantitative‐PCR‐based Detection of Pathogenic Enteric Viruses in Coastal Marine Environment Using Monolithic Chromatographic Supports

Field: Environmental Science (Physical Sciences)

Currently, around 50% of the world population lives in towns and cities within 100 km of the coast. Monitoring of the fecal contamination resulting from anthropogenic activity in coastal water and seawater is essential to ensure the safe use of these water bodies for recreational use, aquaculture, and to assess any biogeochemical changes in the coastal marine environment. The two viruses that are frequently present in contaminated coastal environments, Rotavirus and Norovirus, are also the major cause of human viral gastroenteritis. They are responsible for the manifestation of gastrointestinal disease in nearly 20% of children under the age of 5. Hence, vaccines to control rotavirus-induced gastrointestinal disease have been introduced over the past decade. However, children in the poorest countries account for a majority of rotaviral infection-related deaths, which calls for a balanced approach, where rotavirus vaccination is combined with rapid environmental monitoring of pathogenic enteric virus levels and sanitary management in these vulnerable hotspots. Since exposure to as few as 10–100 particles of enteric viruses is known to cause disease, there is a need to develop a rapid and sensitive diagnostic method. The quantitative PCR (qPCR)-based assays developed can detect as few as 103 enteric viral particles per ml of environmental water samples. However, the detection of these viruses at the levels expected in the coastal marine environment, and the potential presence of PCR inhibitors in these water matrices precludes the use of an initial concentration step. CIM (convective interaction media) methacrylate monolithic columns in conjunction with qPCR assays have been successfully deployed for detection of enteric viral pathogens like rotavirus, norovirus, Hepatitis A and calicivirus from different fresh water, surface water and wastewater matrices. In this study, we evaluate the application of CIM monolithic chromatography to concentrate pathogenic enteric viruses from seawater using rotavirus and norovirus as model viruses. To optimize the concentration of rotavirus, different ion-exchange and hydrophobic interaction chemistries were tested and viral concentration in fractions was quantified by one-step reverse transcription qPCR (RT-qPCR). We present our results on the most effective conditions to concentrate rotavirus at high salinity using CIM-C4 (butyl) hydrophobic interaction monolithic support. Our protocol increases the sensitivity of rotavirus detection by 50–100 fold. The protocol was also successfully deployed in an “on-site” application which would enable the concentration and detection of rotavirus in environmental seawater samples in approximately 5 hours. Furthermore, the extension of our protocols to other enteric viruses, and the scaling-up of the CIM monoliths to handle greater water volumes represents an attractive preparative technique for downstream qPCR and NGS based detection of viral pathogens in the coastal environment. Support or Funding Information This Research and the Postdoctoral Fellowship Awarded to MNB under the Marie Curie Initial Training Network “INTERFACES (Ecohydrological Interfaces as Critical Hotspots for Transformation of Ecosystem Exchange Fluxes and Biogeochemical Cycling)” was Funded by the European Union's Seventh Framework Program for Research, Technological Development and Demonstration under Grant Agreement # 607150