High prevalence of hepatitis E and rat hepatitis E viruses in wastewater in Gothenburg, Sweden

Abstract

Hepatitis E virus (HEV) and Rat Hepatitis E virus (RHEV), recognized for their zoonotic potential, pose significant public health concerns. Our previous research identified both viruses in effluent wastewater in Gothenburg, Sweden. However, there are lingering inquiries regarding the prevalence and genetic diversity of these viruses in influent wastewater, as well as the utility of wastewater surveillance in elucidating their community circulation dynamics. To address these knowledge gaps, we conducted weekly collection of wastewater samples at the Rya wastewater treatment plant in Gothenburg throughout 2023. The concentrations of HEV and RHEV were quantified using quantitative polymerase chain reaction (qPCR). Additionally, two semi/nested-PCR were utilized to amplify viral strains. Furthermore, HEV strains from patients within the same region, as well as other regions in Sweden in 2023, were incorporated into the analysis. Remarkably, we observed a high prevalence of HEV (86%) and RHEV (98%) in wastewater samples, with the majority of HEV sequences identified as subtype 3c/i (9/12). In contrast, HEV subtype 3f was the most sequenced among clinical patient samples (6/12). Notably, previously unreported HEV-3b and unclassified strains were detected in wastewater. Almost all RHEV strains (20/21) were clustered into European groups, with none of the RHEV genetically close to strains previously found in human cases. The notable discordance in prevalence and identified subtypes of HEV-3 in wastewater compared to clinical samples suggests either a significant underdiagnosis of HEV infections or differences in viral loads and shedding durations among humans between HEV-3 subtypes. This underscores the urgent need for improved diagnostic techniques and heightened awareness of HEV transmission dynamics. Furthermore, the consistent detection of RHEV in wastewater underscores the necessity for further investigations to assess the potential role of RHEV in hepatitis cases of unknown etiology, given that most currently available clinical diagnostic assays fail to detect RHEV.

Keywords: HEV-C; Sewage; Subtype; Surveillance; Wastewater-based epidemiology; Zoonotic transmission.

Fig. 1

The concentration of HEV (A) and RHEV (B) in weekly wastewater during 2023.

Fig. 2

Phylogenetic tree of HEV RdRp region. Phylogenetic tree was constructed based on the 339 nucleotides of partial HEV RdRp region using UPGMA method. Red labels represent sequences detected in wastewater samples collected during 2023. Green labels represent sequences isolated from clinical samples within the Gothenburg region during 2023. Blue labels represent sequences isolated from clinical samples from other regions in Sweden. Sequences belonging to subtype 3c/i, 3f, and 3e are highlighted on the right of the phylogenetic tree. The numbers on the tree branches represent bootstrap values, with values greater than 70 shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogenetic tree of RHEV RdRp region. Phylogenetic tree was constructed based on the 378 nucleotides of partial RHEV RdRp region using UPGMA method. Red labels represent sequences detected in wastewater samples collected during 2023 from this study. Green labels represent sequences previously reported associated with human infections. HEV genotypes 1 and 3 were used as outgroup strains. The RHEV sequences are grouped into two major clusters, tentatively named the Asian and European groups. The numbers on the tree branches represent bootstrap values, with values greater than 70 shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Supplementary Fig. 1

Phylogenetic tree of HEV ORF1-ORF2-ORF3 junction region. Only the segments corresponding to the end of ORF1 (positions 4560-5022; NC-001434) were successfully assembled and used for the phylogenetic analysis. Red labels represent sequences detected in wastewater samples collected during 2023 from this study. Sequences belonging to subtype 3c/i and 3b are highlighted on the right of the phylogenetic tree. The numbers on the tree branches represent bootstrap values, with values greater than 70 shown.