Detection and Characterization of Hepatitis E Virus Genotype 3 in Wastewater and Urban Surface Waters in Germany

Abstract

In highly populated areas, environmental surveillance of wastewater and surface waters is a key factor to control the circulation of viruses and risks for public health. Hepatitis E virus (HEV) genotype 3 is considered as an emerging pathogen in industrialized countries. Therefore, this study was carried out to determine the prevalence of HEV in environmental waters in urban and suburban regions in Germany. HEV was monitored in water samples using quantitative RT-PCR (RT-qPCR) and nested RT-PCR without or with virus concentration via polyethylene glycol precipitation or ultracentrifugation. By RT-qPCR, 84-100% of influent samples of wastewater treatment plants were positive for HEV RNA. Genotypes HEV-3c and 3f were identified in wastewater, with HEV-3c being the most prevalent genotype. These data correlate with subtypes identified earlier in patients from the same area. Comparison of wastewater influent and effluent samples revealed a reduction of HEV RNA of about 1 log₁₀ during passage through wastewater treatment plants. In addition, combined sewer overflows (CSOs) after heavy rainfalls were shown to release HEV RNA into surface waters. About 75% of urban river samples taken during these CSO events were positive for HEV RNA by RT-qPCR. In contrast, under normal weather conditions, only around 30% of river samples and 15% of samples from a bathing water located at an urban river were positive for HEV. Median concentrations of HEV RNA of all tested samples at this bathing water were below the limit of detection.

Keywords: Combined sewer overflow; Genotyping; Hepatitis E virus; Monitoring; Surface water; Wastewater.

Fig. 1

Flow chart of methods applied on wastewater and river water samples for HEV RNA detection

Fig. 2

Comparison of HEV RNA concentrations in monthly influent samples (I) and effluent samples (E) of WWTP 1, analysed by RT-qPCR without virus concentration steps. Black and grey bars represent measured HEV concentrations above the LOD (open bars). LOD is the limit of detection with 200 copies/100 ml

Fig. 3

Concentration of HEV RNA in monthly influent samples of an urban WWTP over a period of one year. For comparison of sampling methods direct samples (D) and samples concentrated by ultracentrifugation (U) and PEG precipitation (P) were analysed by RT-qPCR. Black bars represent measured HEV concentrations above the LOD (open bars), which differ in each method

Fig. 4

Characterization of HEV strains from wastewater samples by gel electrophoresis and sequencing. a Exemplary agarose gel with HEV positive samples (332 bp fragments) from two WWTP influent samples. b Maximum likelihood phylogenetic consensus tree of HEV strains detected in urban wastewaters. Numbers at the nodes represent bootstrap values > 60. Scale bar indicates the genetic distance (nucleotide substitutions per site). Identified HEV sequences detected in wastewater samples are marked with a black dot. Names consist of accession numbers, places, months, years of sampling and preparation methods (D: direct sample, U: ultracentrifugation, PEG: polyethylene glycol precipitation). Sequences from HEV infected patients are labelled with open dots. HEV sequences were aligned to 41 HEV-subtype reference sequences denoted by accession number, subgenotype and source of first detection. Since all identified sequences belonged to genotype HEV-3, only sequences of this genotype are shown. Three rabbit HEV-3 sequences were used as outgroup