New Approaches for Enhanced Detection of Enteroviruses from Hawaiian Environmental Waters

Lu Y (2012) New Approaches for Enhanced Detection of Enteroviruses from Hawaiian Environmental Waters. PLoS ONE 7(5): e32442. doi:10.1371/journal.pone.0032442 New Approaches for Enhanced Detection of Enteroviruses from Hawaiian Environmental Waters Christina Connell 0 Hsin-I Tong 0 Zi Wang 0 Erin Allmann 0 Yuanan Lu 0 Niyaz Ahmed, University of Hyderabad, India 0 Departments of Public Health Sciences and Microbiology, University of Hawaii at Manoa , Honolulu, Hawaii , United States of America Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the environment. Reported here are novel and effective laboratory protocols for viral concentration and highly sensitive and optimized RT-PCR for the efficient detection of enteroviruses, an important enteric virus subset, in Hawaiian environmental waters. Eighteen published enterovirus primer pairs were comparatively evaluated for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites tested positive for enterovirus, indicating fecal contamination at these locations. As an additional means of viral concentration, shellfish were collected from 9 sample sites and subjected to dissection, RNA extraction, and subsequent RT-PCR. Shellfish tissue from 6 of 9 sites tested positive for enterovirus. The techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii's environmental waters. - Funding: This work was supported in part by grants from the Centers for Oceans and Human Health (COHH) program, the National Institutes of Environmental Health Sciences (P50ES012740) and the National Science Foundation (OCE04-32479 and OCE09-11000). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Sewage-contaminated recreational water can pose numerous health risks to the public; effective water quality monitoring is therefore absolutely essential [1]. Currently, microbiological water quality is primarily assessed via bacterial indicators such as enterococci, fecal coliform, and total coliform bacteria. However, these indicators often fail to reflect the presence of important hazardous viruses [2]. This is of important concern, as viral pathogens shed in human feces may compromise public safety by polluting recreational waters that meet bacterial indicator standards. Additionally, these bacterial indicators may grow naturally in tropical environments, resulting in inaccurate assessment of water pollution levels [3]. Therefore, alternative monitoring systems are needed to improve the surveillance of recreational waters and secure public protection from waterborne disease [4]. Human enteric viruses, represented by the astroviruses, rotaviruses, noroviruses, adenoviruses, and picornaviruses, have been associated with many waterborne outbreaks and are suggested as alternative indicators of microbial water quality [5,6]. Enteric viruses are primarily transmitted via the fecal-oral route, and viral particles are shed in extremely high numbers from infected individuals [6]. Although most enteric virus infections are primarily associated with diarrhea and self-limiting gastroenteritis, they may also cause hepatitis, conjunctivitis, and respiratory infections. Additionally, in immunocompromised persons, enteric viruses have been associated with aseptic meningitis, encephalitis, and paralysis, all of which have high mortality rates [6]. Common wastewater treatment processes fail to completely inactivate these viruses [7], rendering recreational waters in areas such as Hawaii, where primary-treated sewage is discharged into the sea on a normal basis, vulnerable to viral contamination. Additionally, enteric viruses are able to survive in the environment under a wide pH range and for extended time periods [8]. Due to large viral loads released into sewage-impacted waters, increased environmental persistence compared to indicator bacteria, and the significant role viruses play in waterborne disease, enteric viruses show promising potential to be used as alternative indicators for a more accurate depiction of recreational water quality [6]. This is especially significant in the state of Hawaii, where residents and tourists alike enjoy year-round recreational activities in the local waters. Although the utilization of enteric viruses as alternative water quality indicators is desirable, conventional methods for viral isolation from water are laborious, time-consuming, and inefficient [9]. A major problem encountered is the effective detection of low levels of viruses present in large bodies of water [10]. Because enteric viruses are able to establish infection in humans at low infectious doses, extremely sensitive detection assays are needed. The polymerase chain reaction (PCR) has become an invaluable resource for environmental virologists, favored for its rapidity, sensitivity, specificity, and relative ease-of-use. However, the presence of inhibiting compounds, which can lead to falsenegative results, presents an additional barrier [911]. Detection challenges may be overcome by improved methods for viral concentration from water samples and by efficient inhibitor removal during nucleic acid extraction [11]. Here, we have developed a highly optimized molecular protocol for the effective detection of enteroviruses (EnV) from Hawaiian environmental waters. Enteroviruses, RNA viruses belonging to the Picornavirus family and consisting of coxsackievirus, poliovirus, echovirus, and the numbered enteroviruses, are the most commonly detected enteric viruses in polluted waters and are estimated to cause 30 50 million infections in the US annually [12,13]. The EnV disease spectrum is wide, including gastroenteritis, respiratory infection, diabetes, heart disease, bronchiolitis, conjunctivitis, meningitis, paralysis, and the common cold [6]. Because these viruses are common, fecally shed in extremely high numbers from infected individuals, highly tolerant to salinity and temperature fluctuations, and stable in the environment for extended time periods [8], they have been suggested as a parameter for evaluating viral pollution of environmental waters [13,14]. The availability of permissive cell lines for determining EnV infectivity greatly enh (...truncated)