The Development Of Effective Detection Of Infectious Human Adenovirus From Environmental Watersfor Their Application For Water Quality Monitoring

Background:The current criteria for recreational water quality evaluation are primarily based on measurements of fecal indicator bacteria growth. However, these criteria often fail to predict the presence of waterborne human pathogenic viruses. In the past two decades, illness from recreational water exposure is increasing. EPA (US Environmental Protection Agency) suggests that new alternative indicators are needed for water quality monitoring to ensure recreational water to be safe for people to use since the current used bacterial indicators do not always corresponded with true health risk. Current studies have indicated that human adenovirus could be a potential candidate indicator for fecal contamination because of its relative stability in environmental waters compared to other enteric viruses. Also Human adenovirus has been implicated in some disease outbreaks associated with recreational water exposures. Recently, Our laboratory has established a molecular technique (i.e. PCR) for sensitive detection of human adenoviruses. However, this method has its limitations in differentiating between infectious and non-infectious adenoviruses. In addition, due to lower concentration of enteric viruses in environmental waters and low recovery rate, it has been a major challenge to monitor the presence or absence of infectious adenoviruses in environmental water. In order to overcome these deficiencies and facilitate the use of adenoviruses as a possible indicator for enhanced water monitoring, we tested and established new laboratory methods for effective concentration and recovery of infectious adenovirus from environmental water for viral infectivity test using human adenovirus type7as a model. In addition, we assessed the correlation between infectious viral particles and viral DNA copy numbers by using plaque assay and reat-time PCR, respectively.Methods and results:The present study has shown that the optimal condition for plaque assay of adenovirus type7is by seeding8.0x104/ml of A549cells in12-well plate at1.0mL per well. The PH of virus solution was adjusted to6.4-7.6prior to virus inoculation. Infected cultures were maintained at37?for1.5-2.0hours and overlay medium containing1.6%agarose in DMEM was added at1mL/well. Infected cultures was incubated at37?for9to10days to allow viral plaque development, and then fixed and stained with formaldehyde-crystal violet solution. This optimized plaque assay condition has been used for the entire study to obtain stable and significant results.For effective recovery of concentrated infectious viruses from environmental waters, negatively charged filter membranes (pretreat with AICI3or not) were employed. Five different elution buffers including NaOH, KH2PO4, Glycine, beef extract (BE), and BE with Glycine solution were comparatively tested for their efficiency in recovering infectious adeno viruses from the filter membrane by testing the infectivity of recovered viruses using the established viral plaque assay. Experimnetal tests demonstrated that pretreating the membranes with aluminium chloride prior to filtration and then elution with1mM sodium hydroxide through slowly stirring the membrane for30min are the optimal conditions with the highest virus recovery rate (>80%). To isolate and recovery of infectious adeno virus tissues from affected shellfish-a potential indicator of water contamination, three different viral concentration methods, including using3%beef extract elution-precipitation, PEG precipitation, and ultracentrifugation, were compared. Over90%of infectious viruses were recovered from shellfish tissue homogenates by using3%beef extract precipitation method.To determine the stability of human adeno viruses in environmental waters, A stock of human adenovirus type7was spiked into different experimental conditions including seawater, fresh water, PBS,50%raw sewage,50%sewage effluent,100%raw sewage,100%sewage effluent. It showed that human adenovirus is less stable in seawater than in the other liquid environments. Viral survival curves revealed that human adenoviruses decrease more quickly in effluent than in raw sewage. Also, we can see that the spiked adenovirus declines more quickly in diluted sewage (with50%seawater) than in undiluted sewage.Additionally, to evaluate the bioaccumulation of adenovirus type7in shellfish, Hawaii shellfish species-Isognomonwere collected from ocean and placed into seawater with spiked human adenovirus. These shellfish along with the seawater were monitored for the presence and quantity of adnoviruses at different time points using both plaque assay and real-time PCR. The results indicated that unlike surrounding seawater, shellfish were enabled to accumulate adenovirus to a high concentration(by day5) and retain infectious adenovirus for more than10days. These findings support the possible use of shellfish as a natural bio-indicator for environmental water quality monitoring. Conclusion:This study has established optimized methods for effective concentration and recovery of infectious human adenovirus from environmental waters, and also assessed the stability of human adenovirus in different aquatic environments, which is important for the present consideration of using this virus as an indicator for sewage contamination. This study also supported and warranted future in-depth test of shellfish as a possible and natural indicator for water quality monitoring. Taken together, this research with the establishment of effective and sensitive methods suggests it is possible to use human adenoviruses as an alternative complementary indicator for enhanced monitoring of recreational waters for sewage contamination and also for health risk assessment.