Detection of enteroviruses in marine waters using RT-PCR.

Abstract

Human enteric viruses present in sewage may find their way into recreational marine waters, causing adverse health effects to exposed individuals. This research project focused on (i) optimization of methods for RT-PCR detection of enteroviruses in marine waters, with particular attention to the issue of sensitivity vs inhibition (ii) comparison of enterovirus detection in marine waters by cell culture and RT-PCR and (iii) development of a method for detecting infectious enteroviruses using an integrated cell culture/PCR procedure. Primary sewage effluent and marine water samples were concentrated using Viradel methods and reconcentrated by organic flocculation of beef extract V eluants. Samples were inoculated with poliovirus type 1 (strain LSc-2ab) to evaluate sample inhibition, the effectiveness of column purification and virus recovery efficiency. Samples were subsequently analyzed either by RT-PCR, cell culture or the integrated cell culture/PCR technique. Results from this research indicate: (i) Filterite filters are efficient (66%) for concentration of enteroviruses from marine waters, provided a NaCl and a double BEV elution steps are utilized. (ii) Resin column filtration aids in purifying samples from PCR inhibitory compounds, however, a trade-off exists between inhibition removal and virus recovery efficiencies. (iii) PCR may be used to evaluate large volume concentrates in marine waters, however, due to small sample size and interferences with inhibitory substances, the chance of isolating a virus is still a random chance. (iv) Viruses were isolated using a BGM cell line from primary sewage effluent, a sewage outfall, a canal, and several popular recreational beaches of Hawaii. For comparison, enteroviruses were detected, using RT-PCR, in the sewage, and the outfall only. (v) An integrated cell culture/RT-PCR technique was five times more rapid than traditional cell culture methods for detection of infectious poliovirus in distilled water and enteroviruses in sewage, without the typical problems of PCR inhibitory compounds.