Surveillance for rat hepatitis E in wastewater networks, Italy

ABSTRACT Rat hepevirus is an emerging zoonotic pathogen associated with human hepatitis and distantly related to the hepatitis E virus. By using broadly reactive primers for hepevirus, we detected viral RNA in 68/155 (43.9%) of sewage specimens collected from 14 wastewater treatment plants serving about half of the population of Abruzzo, Italy. On sequence analysis, the 68 strains showed 79.0%–91.6% identities to reference sequences of rodent and human origin, segregating in different genetic groups with a clear geographic/wastewater treatment plant (WWTP)-related pattern. IMPORTANCE Hepatitis E virus (HEV) infection constitutes a significant health problem worldwide. In recent years, in addition to the zoonotic HEV3 and HEV4, emerging highly divergent hepevirus of rat origin (rat HEV [RHEV]) has been associated with human acute and chronic hepatitis. As environmental surveillance can be a complementary tool to explore emerging viruses of human and rodent origin, we investigated the epidemiology and the genetic variability of RHEV targeting 14 wastewater treatment plants in an Italian geographic area considered a hot spot for HEV infection in humans. Our results revealed that RHEV is a significant component of the wastewater microbiota with viral RNA detected in 43.9% of the specimens tested, adding further evidence to the need to investigate more in depth the real burden of RHEV infections in humans.

broad-spectrum PCR assay that detects largely divergent HEV variants (5,6), or metage nomics test based on next-generation sequencing (7) were used in the clinical studies that described the first cases of RHEV infections in humans.
Sewage surveillance has been recognized as a powerful tool to gather information on the epidemiology of infectious diseases in the served population.Untreated wastewater collects viruses excreted by both humans and synanthropic animals, including rodents, thereby providing a comprehensive overview of the viral strains circulating.Herein, to investigate the epidemiology and the genetic variability of RHEV in Italy, we tar geted wastewater treatment plants (WWTPs) of Abruzzo region (southern Italy).This geographic area is considered a hot spot for HEV infection in Italy (8).
The RHEV sequences obtained in this study (GenBank accession numbers OQ930380-OQ930447) shared 82.5%-95.8%nt identity to each other, while identities to all the sequences of rodent and human origin to date available in GenBank were 79.0%-91.6%.On phylogenetic analysis (Fig. 2), the Italian RHEV strains grouped in different genetic clusters with a clear geographic/WWTP-related pattern, a finding that would be consistent with the persistence of the same strain within local rat populations.Interest ingly, the sequence detected in WWTP-6 (Lanciano, Chieti) formed a well-separated subclade (bootstrap value of 86%) with the human RHEV strain recently identified in France (7) from the paraffin-embedded liver biopsy of an immunosuppressed kidneytransplanted patient with cirrhosis.
All the 68 samples containing the sequence of RHEV were screened using a quantita tive RT-PCR (qRT-PCR) specific for HEV, targeting a highly conserved region at the ORF2/ ORF3 overlap, and able to detect the four major HEV genotypes (e.g., Paslahepevirus balayani) (11).However, the qRT-PCR did not detect RHEV RNA, ruling out the presence of mixed infections in the samples and confirming the specificity of the real-time HEV assay (2,6).
Our data revealed that RHEV is a significant component of the wastewater microbiota.This could be accounted for by direct fecal contamination of untreated water by the rodents inhabiting the sewage system.Rodent-associated hepeviruses were first identified in 2010 in fecal and liver specimens from wild rats in Germany (12).Since then, RHEV has been repeatedly detected in several countries in Europe, Asia, and USA, demonstrating a wide distribution in commensal rat populations (Rattus norvegicus, Rattus rattus, and others) (13).Rat species are now recognized as the main host animals of RHEV, and for this reason, it has been hypothesized that close contact with rats or with rat fecal droppings could be the primary transmission route for human infection.Nevertheless, out of the 21 cases of infection worldwide reported thus far (2-7), this    possibility has been deemed plausible only for one patient living in Hong Kong, in a housing estate with evidence of rat infestation in the refuse bins outside his home, while for the other 20 patients, the source of infection remained undetermined.Accordingly, alternative transmission dynamics should be considered.RHEV sequences were recently detected in effluent wastewater at the Rya treatment plant in Gothenburg (Sweden) by NGS (14).The occurrence of RHEV in urban wastewater raises the question as to whether contaminated surface waters can be responsible for waterborne transmission.In Italy, HEV3 RNA has been detected in river waters receiving wastewater discharges and from shellfish, marine waters, and underwater sewage discharges (15,16).Further investiga tions integrating environmental/animal/food surveillance are pivotal to provide a clearer picture of the pathways of RHEV infection.Also, due to the inability of commonly used diagnostic tools to detect RHEV, the global prevalence of infection in the human population is still unknown.Therefore, it will be important to validate specific molecular techniques for RHEV and to include RHEV in the diagnostic algorithm of human acute hepatitis.

FIG 2
The evolutionary history was inferred using the neighbor-joining method.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches.The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site.This analysis involved a total of 104 nucleotide sequences.The sequences detected in this study are in red.

FIG 1
FIG 1 Representation of the geographic map showing the wastewater sampling sites selected in this study from municipal wastewater treatment plants (WWTPs) in Abruzzo (Italy) (A) and list of additional attribute information including the municipality and the number of samples collected (B).The Abruzzo map was created using Quantum GIS (QGIS) software version 3.22; the Italy map came from d-maps.com(https://d-maps.com/carte.php?num_car=4831&lang=en).

TABLE 1
Molecular prevalences and temporal distribution of positive samples for each WWTP in the provinces of Teramo, Pescara, Chieti, and L'Aquila (Abruzzo region, Italy)