Molecular screening of hepatitis E virus in wastewater samples from a swine abattoir

This study aimed to provide novel environmental data on the circulation of hepatitis E virus (HEV) RNA in effluent samples collected from a domestic pig slaughterhouse in Central Italy. Between January and June 2025, a total of 36 samples were sampled in 18 collection days. More specifically, two 1.0 Lt aliquots were taken from two different sites: the first one pre-treatment (V1 - at the stocking yard) and the second one at the post-treatment level [V2 - after the depuration process at the wastewater treatment plant (WWTP)], following the protocol described by dos Santos et al. [1] in Brazil. Both of 18 V1 and V2 samples were centrifuged twice at 180,000 X g for 2 hours; the resulting pellets were used for the RNA extraction procedure using the Trizol LS method. Real time RT-qPCR and nested RT-PCR assays were performed to quantify and amplify the targeting ORF-1 and ORF-2 genetic regions of the HEV genome. Sequencing was followed by the amplicon analysis, publication on the GenBank platform, and phylogenetic tree construction according to the Neighbor-joining method [2]. The IBM SPSS Software (SPSS, Chicago, IL, USA) was used, and the two-tailed t-test was performed to assess possible significant statistical differences in HEV viral loads between V1 and V2 samples, expressed as genomic equivalent per millilitre (GE/mL). Results showed that 13.89% (5/36) of the screened effluent samples detected HEV RNA, and more specifically, 8.33% (3/36) were V1 samples and 5.55% (2/36) V2 ones. The highest viral load (105 GE/mL) was observed in a V1 sample; the average load over the six months was 103 GE/mL. Overall, the t-test revealed statistically significant differences between positive V1 and V2 samples (p-value= 0.040). The amplified ORF-1 and ORF-2 sequences were deposited and published on the GenBank platform with the following accession numbers: PX925942, PX925944, PX925945, PX925946, PX925947, and PX925948. The observed prevalence (13.89%) was higher than 0.4% reported by Rusiñol et al. [3] from effluents collected from a swine abattoir in Spain and lower than 50.0% reported by dos Santos et al. [1] from slaughterhouse wastewaters samples in Brazil. This study represents the first Italian virological and molecular screening of HEV RNA circulation from an environmental perspective. It also confirms the limited efficacy of current WWTPs in removing viral contaminants and highlights the necessity of the inclusion of specific virological parameters in the legislation. These findings want to underline the crucial importance of molecular surveillance of zoonotic viruses among ecosystems.