Figure 1

Figure 1. Emerging rat hepatitis E virus detection in Argentina, 2023–2024. A) Study area in the province of Córdoba (red shading), located in the central region of Argentina, and the location of...

During January 2023–December 2024, we collected sewage samples weekly (total n = 99: 2023, n = 49; 2024, n = 50) from the Bajo Grande wastewater treatment plant (BG-WWTP) of Córdoba city, the capital of the province of Córdoba, with 1,505,250 inhabitants (Figure 1, panel A). BG-WWTP receives the waste of ≈56.9% of the city’s population (https://censo.gob.ar/index.php/mapa_desague_red_publica2). We concentrated each 500-mL sample 100 times by using the polyethylene glycol-6000 precipitation method, as described previously (8), and conducted RNA extraction by using the Nucleic Acid Extraction Versatile Spin Kit (Anatolia Geneworks, https://www.anatoliageneworks.com). We conducted molecular detection of the virus by using a real-time reverse transcription PCR (RT-PCR) targeting a 69-bp fragment of the open reading frame 1 genomic region, as described previously (10). We used the TaqMan Fast Virus 1-Step master mix (Thermo Fisher Scientific, https://www.thermofisher.com), performed in a StepOne Real-Time PCR (Thermo Fisher Scientific). For positive controls we used synthetic oligonucleotide (Appendix), and for negative controls we used sterile water. We considered samples RNA R-HEV positive if we observed an S-shaped curve in the specific detection channel of each specimen and obtained a cycle threshold value <40 for viral specific target, as previously described (1,8). With the aim of genetic characterization of positive samples, we conducted heminested RT-PCR amplification of a 338-bp fragment within the open reading frame 1 genomic region of the Hepeviridae family (11). We purified PCR products by using the PureLink Quick Gel Extraction Kit (Invitrogen, https://www.invitrogen.com) and sequenced in both directions by using an Applied Biosystem 3500XL Genetic Analyzer (Thermo Fisher Scientific). We conducted phylogenetic analyses by using MEGA version11 (12), IQ-Tree (http://iqtree.cibiv.univie.ac.at), and FigTree (https://tree.bio.ed.ac.uk/software/figtree). In addition, we used a newly developed genotyping tool for R-HEV classification (13) and constructed an identity matrix to determine the similarity between the sequences obtained by using BioEdit software version 7.7.1 (https://bioedit.software.informer.com/Descargar-gratis).

Figure 2

Figure 2. Maximum-likelihood phylogenetic tree on the basis of a 338 bp fragment of the open reading frame 1 genomic region of rat hepatitis E virus (R-HEV; Rocahepevirus ratti) from...

Of the 99 samples analyzed, 67.7% (95% CI 58.5%–76.9%; n = 67) tested positive for R-HEV detection by RT-PCR (Appendix Table 1). By year, 77.6% ; (95% CI 63.0%–87.8%; n = 38) of samples from 2023 were positive, and 58.0% (95% CI 43.3%–71.5%; n = 29) of samples from 2024 were positive (Figure 1, panel B). Of the 67 positive samples by real-time RT-PCR, 14 were positive by the heminested RT-PCR, and 10 were sequenced. The phylogenetic tree confirmed the virus species identity as Rocahepevirus ratti (GenBank accession nos. PX060496–504, PX060508) and showed grouping with sequences from sewage samples from Italy and rodents from Canada, within genotype C1 (Figure 2). The genotyping tool indicated that 9 sequences from this study belonged to the proposed clade I, subtype a. One sequence yielded an indeterminate result (Appendix Table 2). The similarity between the obtained sequences varied from 0.71 to 0.98 (Appendix Table 3).

This study revealed R-HEV circulation in Argentina, confirming the presence of emerging R-HEV in South America. R-HEV detection in wastewater samples shows a high level of circulation (high detection rate) in the human environment from the central region of the country. The low number of sequenced samples could be explained by the dilution and degradation of viral RNA within the wastewater matrix, driven by factors such as pH fluctuations, temperature variation, and complex biochemical interactions. Those conditions might promote the formation of short, partially degraded RNA fragments that remain detectable by real-time RT-PCR but are insufficient for amplifying longer genomic regions required for conventional PCR and sequencing. Similar patterns were reported in previous wastewater studies (8). Contamination of wastewater by human waste is a possible source of the virus, although the involvement of rodents, the natural viral host, in sewers is the most likely scenario (14). To date, we have not found evidence of human or animal infections in Argentina; therefore, our results open new and unexplored fields for R-HEV research in South America. Studies from the past 2 years have demonstrated the presence of R-HEV RNA in wastewater from countries in Europe, underscoring the value of wastewater-based surveillance for detecting emerging viruses such as R-HEV (1,8). Concomitantly, studies were conducted to clarify the clinical implications of R-HEV in Europe. Those studies revealed that R-HEV could cause symptomatic disease in humans, both in immunosuppressed and immunocompetent patients, producing mild illness, severe hepatitis, and potentially death (4).

Phylogenetic analysis revealed that the sequences obtained in our study clustered together, although they were not identical (similarity 0.71–0.98). That clustering could suggest that the same strains circulated over the 2-year period. The sequences grouped close to strains previously detected in sewers in Italy and in rodents from Canada within genotype C1 (1,15). However, because of the limited available data—very few sequences of this virus are currently deposited in GenBank, and 3 are from South America—and because of the relatively short genomic fragment analyzed, definitive conclusions cannot be drawn. Recently published studies suggest our sequences would be assigned to clade I, subtype a (13).

In line with the One Health approach, our findings reinforce the need to further investigate R-HEV circulation in Argentina and South America. This effort should integrate environmental surveillance with clinical research, such as testing samples from symptomatic patients for R-HEV RNA and exploring potential viral reservoirs such as rats. In addition to rodents, attention should be given to other possible hosts, such as pigs, in which R-HEV infections have been documented (6).