New approaches for enhanced detection of enteroviruses from Hawaiian environmental waters

Abstract

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.

Figure 1. Environmental sampling sites around the island of O’ahu, Hawaii.

Figure 2. Agarose gel depicting enterovirus detection from urban sewage.

Amplified with primer set EQ-1/EQ-2. Detection from 100 mL of raw influent, post-primary clarification/pre-UV disinfection, and post-disinfection/effluent treatment stages. M = 50 bp DNA ladder. (-) = no template control.

Figure 3. Nucleotide sequence analysis of EnV isolated from wastewater (multiple clones), water, and shellfish samples.

(A) Sequence alignment of 142 bp fragments amplified by primer set EQ-1/EQ-2. Dots indicate homology with sewage isolate #1. (B) Closest BLAST match (including E value and percentage identity) of sequenced PCR products with EnV strains listed in the NCBI database.