Development of optimal methods for effective detection and recovery of infectious enteric viruses from the environmental water

Abstract

Human enteric viruses have been used as alternative indicators for water quality monitoring since the current used bacterial indicators do not always corresponded with true health risk. Effective laboratory methods for viral detection and concentration are essential since these human enteric pathogens exist in an extremely low number in environmental water. Our laboratory has recently established a molecular technique, i.e. PCR, for effective concentration and sensitive detection of these viruses. However, this method has limitations for differentiating between infectious and non-infectious viruses. Therefore, this study is aimed to establish optimized laboratory conditions for concentration and recovery of infectious viruses from environmental waters. For effective recovery of concentrated infectious viruses from negatively charged filter membranes, three different elution buffers: NaOH, KH2PO4 and beef extract (BE) solution were compared. And for viral isolation and infectivity from shellfish tissues, PEG precipitation method and ultracentrifugation method were compared by using Poliovirus type 1 as a viral model. Viral infectivity was conducted through plaque formation assay and demonstrated that 3% BE in 50 mM glycine is the most effective buffer for virus recovery (>90%). And over 77% of infectious viruses were recovered from shellfish tissue homogenates through PEG 6000 precipitation. The established viral plaque assay was also used to determine the stability of spiked Poliovirus 1 under various experimental conditions including seawater, fresh water, PBS, 50% raw sewage, 50% sewage effluent, 100% raw sewage, 100% sewage effluent and also in the indigenous bivalve mollusk tissue. It showed that Poliovirus 1 is less stable in sea water than the other aquatic environments. Viral survival curves revealed no significant difference in the stability of infectious Poliovirus 1 between raw sewage and sewage effluent. However, they illustrated that Poliovirus 1 is more stable in 100% raw sewage/sewage effluent than it in 50% raw sewage/sewage effluent. Additionally, the indigenous shellfish (isognomon spp.) was observed to have an accumulation function for infectious Poliovirus 1 in their tissue and could retain the virus for a longer time than in the surrounding water which enhanced the use of shellfish as a natural bio-indicator for environmental water quality monitoring. This study has established optimized laboratory conditions for the concentration and recovery of infectious enteric virus and the determination of viral stability in different environments. This provides the essential information for the facilitation of current tests using enteric viruses and shellfish as indicators for aquatic environmental monitoring and for assessing contamination-associated health risks.