Determining the relationship of human enteric viruses in clinical, wastewater, and environmental samples utilizing molecular and cell culture techniques

Jacquelina Susann Woods

Abstract

This study was the first to examine five significant enteric viruses in human fecal material, sewage, and oysters to show a genetic relationship between human enteric viruses and different sample matrices. Fecal samples were collected from an area hospital and examined for norovirus genotype I (NoV GI), norovirus genotype II (NoV GII), hepatitis A virus (HAV), adenovirus (ADV), and enteroviruses. During this study, sewage samples were collected from a Waster Water Treatment Plant (WWTP) in Mobile, AL and oyster sentinels were placed at 0.1 nautical miles (nm) (station 1), 0.2nm (station 2), 1.5nm (station 3), and 4nm (station 4) downstream from the WWTP. Samples were examined by molecular methods for the five virus groups; HuAdv, HAV, and enteroviruses were examined by cell culture methods. Samples positive by molecular methods were further examined by sequencing PCR products of NoV, HuAdv, and enteroviruses. Of the 401 fecal samples analyzed, human NoV, HuAdv, and enterovirus was detected in 4.7%, 13.8%, and 2.5% of samples respectively. HAV was not detected in any fecal, oyster, sewage, or tissues culture samples. HuAdv was detected in the sewage influent and effluent and station 1 in all samples tested during the study. Enterovirus was detected in 5 out of 7 of influent sample sets and 1 out of 7 of oyster concentrates. The detection rate for viruses in oysters placed at stations 1 and 2 were similar for all viruses tested including male-specific bacteriophage (MSB). Sequence analysis of NoV GII for the September and December sample set revealed ≥99% sequence homology for stool isolates and oyster isolates at station 2. Sequence analysis for HuAdv for the December samples revealed ≥99% sequence homology between the influent, oyster isolates, and tissues culture isolates. NoV GII and HuAdv were detected at all stations during December sampling utilizing real time PCR and RT-PCR, respectively. NoV GII was detected at all stations by conventional RT-PCR for the February samples at all stations. This study showed that there is significant genetic relatedness between clinical and environmental isolates.