
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39193634
2396865
10.1080/22221751.2024.2396865
Version of Record
Hepatitis-Updates from the Classical Emerging Infection
Research Article
Serological and molecular survey of rat hepatitis E virus (Rocahepevirus ratti) in drug users
EMERGING MICROBES & INFECTIONS
M. CASARES-JIMENEZ ET AL.
Casares-Jimenez Maria ab
Corona-Mata Diana ab
Garcia-Garcia Transito c
Manchado-Lopez Leticia d
Rios-Muñoz Lucia a
de Guia-Castro Maria d
Lopez-Lopez Pedro ab
Caceres-Anillo David d
Camacho Angela ab
Caballero-Gomez Javier abe
Perez-Valero Ignacio ab
Gallo-Marin Marina a
https://orcid.org/0000-0001-9655-817X
Perez Ana Belen bf
Ulrich Rainer G. gh
https://orcid.org/0000-0001-5740-6675
Rivero-Juarez Antonio ab
Rivero Antonio ab
a Grupo de Virología Clínica y Zoonosis, Unidad de Enfermedades Infecciosas, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Universidad de Córdoba (UCO), Córdoba, España
b CIBERINFEC, ISCIII-CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III. Majadahonda, Madrid, España
c Immunogenomic and Molecular Pathogenesis, Zoonoses and Emerging Diseases Unit (ENZOEM), Genetic Department, University of Cordoba, Cordoba, Spain
d Unidad de Drogas y Adicciones-CPD (UDA-CPD), Instituto Provincial Bienestar Social, Diputación Córdoba, Córdoba, España
e Departamento de Sanidad Animal, Grupo de Investigación en Sanidad Animal y Zoonosis (GISAZ), UIC Zoonosis y Enfermedades Emergentes (ENZOEM), Universidad de Córdoba, Córdoba, España
f Unidad de Microbiología, Hospital Universitario Reina Sofía, Córdoba, España
g Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany
h German Centre for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Greifswald-Insel Riems, Germany
CONTACT Antonio Rivero-Juarez arjvet@gmail.com Grupo de Virología Clínica y Zoonosis, Unidad de Enfermedades Infecciosas, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Universidad de Córdoba (UCO), Avenida Menéndez Pidal, s/n., 14004 Córdoba, España
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© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Rat hepatitis E virus (ratHEV) is an emerging cause of acute hepatitis of zoonotic origin. Since seroprevalence studies are scarce, at-risk groups are almost unknown. Because blood-borne infections frequently occur in people with drug use, who are particularly vulnerable to infection due to lack of housing and homelessness, this population constitutes a priority in which ratHEV infection should be evaluated. Therefore, the aim of this study was to evaluate the ratHEV seroprevalence and RNA detection rate in drug users as a potential at-risk population. We designed a retrospective study involving individuals that attended drug rehabilitation centres. Exposure to ratHEV was assessed by specific antibody detection using ELISA and dot blot (DB) assay and the presence of active infection by ratHEV RNA detection using RT-qPCR. Three-hundred and forty-one individuals were included, the most of them being men (67.7%) with an average age of 45 years. A total of 17 individuals showed specific IgG antibodies against ratHEV (4.6%; 95% CI; 3.1%–7.9%). One case of active ratHEV infection was identified (0.3%; 95% CI: 0.1%–1.8%). This was a 57-year-old homeless woman with limited financial resources, who had active cocaine and heroin use via parenteral route. In conclusion, we identified a potential exposure to ratHEV among drug users. Targeted studies in drug users with proper control groups are necessary to evaluate high-risk populations and transmission routes more accurately.

KEYWORDS

Rat hepatitis E virus
hepatitis E
Zoonoses
drug users
public health
Andalusian General Secretariat for Research, Development, and Innovation in Health PI-0287-2019 Spanish Ministry of Health RD12/0017/0012 European Regional Development Fund (ERDF) Carlos III Health Institute (Research Project Spanish Junta de Andalucía C1-0001-2023 DZIF Thematic Translational Unit This work was supported by the Andalusian General Secretariat for Research, Development, and Innovation in Health (PI-0287-2019), the Spanish Ministry of Health (RD12/0017/0012), co-financed by European Regional Development Fund (ERDF), and the Carlos III Health Institute (Research Project [grant numbers: PI21/00793 and PI22/01098]. Projects “PI21/00793” and “PI22/01098” were funded by Carlos III Health Institute (ISCIII) and co-funded by the European Union. ARJ is supported by a contract from the Spanish Junta de Andalucía (Nicolas Monardes program: C1-0001-2023). JCG is supported by the CIBERINFEC (CB21/13/00083), Carlos III Health Institute, Spanish Ministry of Science and NextGenerationEU. MCJ is the recipient of a PFIS predoctoral grant (FI22/00180) from the Carlos III Health Institute and co-funded by the European Union. LRM is the recipient of a “INVESTIGO” research programme grant funded by the European Union NextGenerationEU Plan. DCM is the recipient of a “Rio-Hortega” (CM22/00176) grant from the Carlos III Health Institute and co-funded by the European Union. MG and PLL were supported by postdoctoral contracts Margarita Salas (University of Murcia and University of Córdoba, respectively) from the Program of Requalification of the Spanish University System (Spanish Ministry of Universities) financed by the European Union-NextGenerationEU. JCG is supported by the CIBERINFEC (CB21/13/00083), Carlos III Health Institute, Spanish Ministry of Science and NextGenerationEU. TGG is recipient of a “Ramon y Cajal” contract funded by MCIN/AEI/10.13039/501100011033 and NextGeneration EU/PRTR. The laboratory of RGU is supported by DZIF Thematic Translational Unit (TTU) “Emerging Infections.” (grant number 01.808; awarded to RGU).
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pmcIntroduction

Rat hepatitis E virus (ratHEV, species Rocahepevirus ratti) is a widespread pathogen found in urban rats across Asia and Europe [1]. It was not considered to have zoonotic potential because of its genetic divergence to human pathogenic hepatitis E virus (HEV, species Paslahepevirus balayani) and its inability to infect nonhuman primates [2], until 2018, when the first human case was reported in a liver transplant recipient in Hong Kong [3]. Subsequently, more cases of ratHEV infection were reported in Canada [4], Spain [5], and the most recent one in France, a chronic ratHEV infection associated with cirrhosis in an immunosuppressed patient [6]. Despite the transmission route is unknown, we speculate that humans may potentially acquire the infection through direct contact with surfaces contaminated by rat droppings or indirectly through contaminated water or food products. The transmission of ratHEV to humans through intermediate animal hosts (e.g. domestic animals [7], etc.) or through blood transfusion and/or organ transplantation remains an open question [8, 9].

Because of the low number of cases reported, risk factors associated with ratHEV infection are unknown, therefore, it's not possible to establish a high-risk population and, consequently, preventive measures cannot be recommended. The definition of potential high-risk populations needs to be done based on the current knowledge of ratHEV epidemiology. In this sense, individuals with strong contact with urban rodents (risk for direct transmission), living in poor sanitation conditions (risk for indirect transmission), and with an increased risk for blood-borne transmission of the pathogen, might represent key populations to evaluate the occurrence and frequency of infections with this emerging virus. Blood-borne infections frequently occur in drug users linked to high-risk behaviours like sharing needles [9]. Moreover, they are particularly vulnerable to infection due to lack of housing and homelessness [10]. Therefore, people with substance abuse constitute a priority population in which ratHEV should be evaluated.

Methods

Study concept and design

This was a retrospective study including individuals attending twelve drug and addiction centres (DAC) from the city of Cordoba (Andalusia, southern Spain. 325,708 inhabitants), between April 2019 and July 2020. These centres target all patients with drug or toxic substance dependence in the city. The strategy of recruitment and follow-up of this population has been described elsewhere [11]. Criteria for people to be included in the present study were: (i) older than 18 years, and (ii) serum sample available in the Andalusian Health System Biobank.

Antibodies evaluation for HEV and ratHEV

All patients were tested for specific immunoglobulin G (IgG) using a dot blot (DB) assay which was able to identify IgG antibodies reacting to HEV genotype 3 (HEV-3) and ratHEV. The capability of this assay to investigate ratHEV and HEV-specific IgG antibodies was previously verified after testing a set of serum samples including patients with acute hepatitis caused by HEV, ratHEV, and other aetiologies, and tested in a large cohort of people living with HIV [12]. Briefly, His-tagged and affinity-purified carboxy-terminal segments of Escherichia coli (E. coli)-expressed capsid proteins (Ctr) served as antigens for HEV-3 and ratHEV as previously described [13]. For the negative substrate control (substrate control 1), we used a nucleocapsid protein derivative obtained from the Puumala orthohantavirus strain Vranica/Hällnäs, which has been described to have different epitopes specific for the acute IgG response in humans [14]. The hantavirus protein was used in a comparable expression system (E. coli; His-tag fusion) so the hantavirus protein represents an antigen that have the same structure with an N-terminal His-tag as the HEV capsid protein derivatives. To exclude a reactivity of this hantavirus-derived protein with sera from hantavirus-infected patients, the immunodominant region was deleted (aa residues 40-212). Meanwhile, our positive substrate control (substrate control 2) consisted of an E. coli M15pREP4 lysate. This lysate originates from a bacterial culture of M15 cells containing the pREP4 plasmid, which grants resistance to kanamycin and continuously expresses the lac repressor protein encoded by the lac I gene. The bacterial cell lysate serves as a positive substrate control in our study due to its ability to detect a natural antibody response against E. coli proteins. E. coli is a ubiquitous bacterium commonly found in the intestinal microbiota of healthy individuals. As a result, antibodies targeting E. coli proteins are naturally present in the sera of healthy patients. This approach allows us to use an assay by ensuring that it can reliably detect antibodies that are naturally present in healthy individuals due to exposure to common bacteria like E. coli. The use of this lysate as positive substrate control has been widely accepted [15-17].

Before diving into the experimental analyses, thorough validation steps were undertaken to ensure the accuracy and reliability of the results. These steps involved meticulous control blots with monoclonal antibodies and subsequent testing with polyclonal human sera. Firstly, control blots with monoclonal antibodies were conducted to scrutinize the reactivity of the purified proteins and lysates. To evaluate the reactivity of the antibodies with various antigens, an anti-His-tag monoclonal antibody blot was first employed. This analysis was conducted with all His-tagged antigens except for the M15pREP4 total E. coli lysate, which served as a negative control here. Following this, an anti-HEV blot was utilized to specifically target HEV and ratHEV antigens. For this purpose, a monoclonal antibody was used that was raised against HEV-3 Ctr protein, but reacts with proteins of ratHEV, but also HEV-1, HEV-2, HEV-4 and HEV-7 [18]. It was crucial to ensure that this reaction did not extend to the hantavirus antigen, thereby confirming the specificity of the antibodies used. Finally, an essential control was performed with an anti-hantavirus blot. This blot was designed to exclusively detect the hantavirus antigen without any cross-reactivity with HEV antigens. This step was vital to distinguish between the different antigens and to prevent any misleading interpretations. The experimental analysis then advanced to the blot with polyclonal human sera. Purified HEV antigens and the hantavirus antigen were exposed to polyclonal human sera to evaluate their reactivity. This step aimed to determine whether the immune response was cross-reactive or specific to selected antigens. The inclusion of purified hantavirus antigen as a negative control was crucial. Any reactivity observed with this control would indicate non-specific binding, prompting a reevaluation of the assay's reliability.

For DB analysis, 5 µl of each purified protein was spotted onto an activated polyvinylidene difluoride (PVDF) membrane. 2 µl of each human serum sample was used as the primary antibody, incubated overnight, and the antigen–antibody reaction was detected by adding purified recombinant protein A/G conjugated with horseradish peroxidase (HRP) (Thermo Scientific, Schwerte, Germany). Exposure to ratHEV was confirmed when a reactive dot was observed against ratHEV antigen, while no such observation was made for HEV-3 and the substrate control 1. Exposure to HEV was confirmed when a reactive dot was observed against HEV-3 antigen, while no such observation was made for ratHEV and the substrate control 1. A cross-reactive antibody response was determined by a reactivity of the human serum with HEV-3 and ratHEV antigens, but lacking reactivity with the substrate control 1 antigen. This cross-reactive antibody response is considered as indeterminate result due to the exposure to both viruses or a cross-reactive antibody response. If there was no reactivity to HEV-3, ratHEV and substrate control 1, the blot was considered to be negative (Figure 1). If a patient showed a negative result to E. coli M15pREP4 substrate control 2, the result was considered “not evaluable.” Additionally, we aimed to evaluate the efficacy of a strategy involving the testing of DB samples for specific ratHEV IgG antibodies exclusively in individuals who exhibit IgG antibodies to HEV by enzyme-linked immunosorbent assay (ELISA). To assess the performance of this approach, we conducted analyses on all samples for ratHEV and HEV-3 antigens by DB and for HEV IgG by ELISA using the Wantai Diagnostic IgG kit (Beijing, China) [19]. Figure 1. DB results for sera that tested positive for ratHEV-specific IgG antibodies. Serum sample set of patients suffering for acute hepatitis E virus infection (n = 3), patients with acute ratHEV infection (n = 3) and patients with acute hepatitis of unknown origin (n = 5) were analyzed as control of the assay used (Figure 1(A)). Individuals included in the study with detection of ratHEV-specific IgG antibodies according to ELISA evaluation status (Figure 1(B)). The numbers correspond to the sample’s identification. His-tagged and affinity-purified carboxy-terminal segments of Escherichia coli (E. coli)-expressed capsid proteins served as antigens for HEV-3 and ratHEV (HEV and ratHEV). As negative substrate control (SC1 (-)), a his-tagged E. coli-expressed and affinity purified nucleocapsid protein derivative of the Puumala orthohantavirus strain Vranica/Hällnäs was used. As positive substrate control (SC2 (+)), an E. coli M15pREP4 total lysate was used.

Molecular evaluation for ratHEV infection

In all individuals ratHEV RNA was evaluated by RT-qPCR. Total RNA was extracted from 400 µl of serum using the QIAamp Mini Elute virus spin kit (Qiagen, Hilden, Germany) using an automated procedure (QIAcube. Qiagen, Hilden, Germany) and purified RNA was eluted in a 50 µl volume. An in-house RT-qPCR protocol targeting open reading frame 1 (ORF1) of ratHEV was used [20], using the One Step Script III RT‒PCR Kit (Takara Bioscience. Kyoto, Japan). As a positive control, RNA from a rodent liver sample that previously tested positive in our laboratory was employed (GenBank accession number: OR282813). Positive samples were re-amplified by a conventional RT–PCR and sequenced following a protocol targeting 880 nucleotides (nt) located in the ORF1 region [21]. Amplicons were examined on 1.5% agarose gels stained with RedSafeTM Nucleic Acid Staining solution (iNtRON Biotechnology, Seongnam, Korea). RT-qPCR products with the correct target size were purified using Illustra™ ExoProStar™. Both sense strands were sequenced using a BigDye Terminator cycle sequencing ready reaction kit on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA). For taxonomic assignment within the Hepeviridae family, we used the HEVnet genotyping tool (https://www.rivm.nl/mpf/typingtool/hev/) and confirmed the results by Basic Local Alignment Search Tool (BLAST) analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analysis

The presence of ratHEV-specific IgG antibodies detected by DB assay was taken as the main outcome variable. The secondary outcome variable was the presence of ratHEV RNA. The seroprevalence of ratHEV was calculated, providing a two-sided 95% confidence interval (95% CI) using the exact binomial distribution. Variables were expressed as number of cases (%) or median (IQR).

Ethical statement

This study was designed and performed according to the Helsinki Declaration. The Andalusian Ethical Committee approved the study protocol (PEIBA_5081 minutes 325 of June 29th, 2021). Samples were collected and cryopreserved at -80°C in the Andalusian Health System Biobank (National Registry Reference: B.0001601).

Results

Study population

A total of 341 patients fulfilled the inclusion criteria and were included in the study. Main population baseline characteristics are shown in Table 1. Most of the tested population were men (84.9%) with a median age of 45 years. The 17.3% of the population actively consumed heroin by injection. Thirty-eight individuals (11.1%) were infected by hepatitis C virus (HCV) and three people (0.9%) lived with human immunodeficiency virus (HIV). None of the individuals showed active hepatitis B virus (HBV) infection. Table 1. Demographic characteristics of patients included in the study.

Variable	Global
(n = 341)	ratHEV IgG + (n = 17)	ratHEV IgG-
(n = 324)	P	
Gender, n (%)	 	 	 	 	
Female, n (%)	48 (14.8)	5 (29.4)	53 (15.5)	0.265	
Male, n (%)	275 (84.9)	12 (70.6)	287 (84.2)	 	
Age (years), median (Q1–Q3)	45 (37–54)	55 (42–59.5)	44.5 (37–52)	0.05	
HCV infection, n (%)	38 (11.1)	2 (11.8)	36 (11.1)	0.992	
HIV infection, n (%)	3 (0.9)	0 (0)	3 (0.9)	0.905	
Drug use, n (%)	 	 	 	 	
Cocaine	148 (43.8)	5 (29.4)	143 (44.1)	0.443	
Heroin	59 (17.3)	3 (17.6)	56 (17.3)	0.602	
Alcohol	219 (64.2)	9 (52.9)	210 (64.8)	0.057	
Cannabis	109 (32)	4 (23.5)	105 (32.4)	0.251	
Other	12 (3.5)	3 (17.6)	9 (2.7)	0.461	
Methadone	5 (1.5)	1 (5.9)	4 (1.2)	 	
Opioids	2 (0.6)	0	2 (0.6)	 	
Benzodiazepines	4 (1.2)	2 (11.8)	2 (0.6)	 	
Non-substance related	1 (0.3)	0	1 (0.3)	 	
Legend: Rat hepatitis E virus (ratHEV); number of cases (n); hepatitis C virus (HCV); human immunodeficiency virus (HIV).

Serological evaluation

A total of 17 patients showed specific IgG antibodies for ratHEV by DB assay supposing a seroprevalence of 5% (95% CI; 2.8%−7.2%) (Figure 1). The characteristics of patients exhibiting ratHEV-reactive antibodies are shown in Table 1. All variables were similar between patients with specific ratHEV-specific IgG antibodies and those not. Nevertheless, patients with specific ratHEV-specific IgG antibodies were older than negative individuals (p = 0.005).

Eighty-one individuals (23.8%; 95% CI: 19.5% – 28.6%) were reactive to anti-HEV IgG antibodies by ELISA. Of the 17 individuals showing ratHEV-specific IgG antibodies, only four (1.2% (95% CI; 0.3% – 3.1%)) were also positive for anti-HEV IgG by ELISA. Thus, most individuals bearing ratHEV-specific IgG antibodies could not be detected if a previous screening was done by ELISA (3.8% (95% CI; 2.2%–6.5%)). Six individuals (1.8% (95% CI; 0.7%–3.9%)) showed cross-reactivity for both HEV and ratHEV antigens. Fifty-one individuals (15% (95% CI; 11.5%–19.2%)) were reactive to anti-HEV IgG by DB, where 7 (2.1% (95% CI; 0.9%–4.3%)) of them, were not detected by the Wantai IgG HEV ELISA. All the results obtained by DB and ELISA are shown in Table 2. Table 2. Individuals showing ratHEV specific IgG antibodies and HEV specific IgG antibodies by DB and commercial HEV IgG ELISA.

 	HEV DB	ratHEV DB	Cross-reactive	Negative	Total	
Wantai IgG+	44	4	4	29	81	
Wantai IgG-	7	13	2	238	260	
Total	51	17	6	267	341	
Legend: Hepatitis E virus (HEV); rat hepatitis E virus (ratHEV); dotblot (DB).

Molecular evaluation of ratHEV

One individual was positive for ratHEV RNA supposing a prevalence of infection of 0.3% (95% CI; 0.1%–1.8%). The sequence analysis confirms a ratHEV strain (GenBank accession number: PP256210) with a sequence similarity ranging between 87% and 88.6% with other strains previously identified in rats from Spain and the study area [22] (Figure 2). This individual was a homeless 57-year-old woman with limited financial resources and active abuse of alcohol, cannabis, cocaine, and injected heroin. She previously underwent treatment for psychoactive substance dependence with methadone. She has been diagnosed with depressive disorders. At sample collection, she showed normal level on alanine transaminase (ALT) and without clinical symptoms. Serology results indicate that she was negative for HCV and HIV. Concerning HBV serological markers, she exhibited positive Anti-HBs, positive Anti-HBc, and negative HBsAg, indicating past infection. She was negative for HEV-specific IgG and IgM antibodies by ELISA and negative for ratHEV-specific IgG antibodies. Figure 2. Phylogenetic analysis of the ratHEV sequence identified in the study. Sequence of the patient identified in the present study is marked with a circle (•). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The analysis involved 70 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There are a total of 770 positions in the final dataset.

Discussion

RatHEV represents a recent emerging virus of significant importance to public health, not only in Asia but also in Europe and Northern America. Currently, it has not been clearly identified which populations are at higher infection risk, making the implementation of specific preventive measures difficult. To address this challenge, it is necessary to develop and apply serological assays that allow differentiation between antibodies specific for ratHEV to those specific for HEV-3 or other hepeviruses. This challenge is increasing by the discovery of further hepeviruses in small mammals including those with synanthropic behaviour [23]. Our study shows that almost 5% of drug users exhibited specific antibodies against ratHEV, revealing that exposure to ratHEV is more common than initially expected. Based on this finding, as well as the high percentage of rats testing positive in different studies [22], as well as the identification of seropositive cases in humans across several regions of the Spanish territory, ratHEV should be ranked as a significant public health concern in Spain. Given the similarity in socio-economic characteristics between Spain and other European countries, it is reasonable to consider that the evidence from Spain could be extrapolated to the rest of the European continent.

Our study was not designed to assess attendees at DACs as a high-risk population because a control/comparative group is not included. Despite that, an indirect comparison might be done with these previous serosurveys conducted in different populations from Asia and Europe. The highest seroprevalence was attained in a study conducted in Vietnam, where hospitalized febrile individuals were tested by a specific ratHEV ELISA [24]. In this study, a seroprevalence of 47.5% was reached, suggesting a wide and common exposure to ratHEV among in this setting. This contrasts with the other available studies where a seroprevalence around 1% was obtained. In this sense, a recent study in Hong Kong found a seroprevalence of ratHEV among solid organ transplant and immunocompetent donors of 1.2% and 0.7%, respectively [25]. In the same way, the two studies conducted in Europe reported a seroprevalence of 1.9% in forestry workers and 1.1% in people living with HIV [26, 22]. Our study reports a seroprevalence of 5% in drug user, constituting the highest seroprevalence reached in European´ population. Although studies cannot be directly compared, our results could suggest that conditions surrounding drug abuse could favour the exposition to ratHEV. In this sense, from an epidemiological perspective, given the circumstances of precarious hygiene, limited resources, and frequent homelessness, individuals may have come into contact with rats or their droppings, potentially leading to infection. Furthermore, the case of active infection identified in our study was a woman who was actively consuming heroin intravenously. Addictive injection behaviour often correlates with the spread of viral hepatitis, and hepatitis outbreaks have been frequently reported among the homeless, who are associated with low educational level and poor hygiene conditions that can favour transmission [27, 28]. Studies including a control group are needed to evaluate this possible association as well transmission routes among drug users need to be further investigated.

The connection between other hepeviruses, such as HEV, and drug consumption through intravenous injection is a topic that is not yet fully understood and generates controversy. Meanwhile, there are studies which found similar HEV seroprevalence between intravenous drug users and the general population [29], whereas other studies found a higher trend in this vulnerable population [10, 30]. Despite the contradictory results, the only study with a proper design to evaluate this possible association, including an age-and sex-matched organ donor control group, report that parenterally consume drugs had a higher HEV IgG seroprevalence (46.2% in the parenteral drug users vs. 22% in the control group, p < 0.001) [10]. On the other hand, any of the consumed drugs were found to be associated as a risk factor for occurrence of ratHEV-specific antibodies. The only factor associated with ratHEV infection was age, where ratHEV-seropositive individuals seem to be older than those not. This factor was also associated with HEV seroprevalence in other studies. In a study conducted on a population of individuals who use crack cocaine, the results showed a higher seroprevalence and an increased likelihood of presenting antibodies against HEV in individuals over 35 years old [31]. According to this finding, ratHEV is similar to HEV in this aspect.

The serological assessment of ratHEV is not yet fully established and is in a limited stage of development. Currently, there is no commercial kit for the detection of ratHEV-specific antibodies, with only in-house assays available [24, 25, 26, 22]. The assay used in our study, despite the verification of the capability of our assay to detect ratHEV-specific IgG antibodies, it might be difficult to implement it in large population screenings because the intrinsic characteristics of an in-house assay. Therefore, the exploitation of the cross-reactivity in a commercial screening IgG ELISA kit, as used in our study [32], might be an efficient strategy to combine the rapid high-throughput cross-reactivity screening and the subsequent serotyping, e.g. by the DB described here. Our study shows, however, that this strategy is not effective in identifying individuals with ratHEV specific IgG. In this way, approximately 3 out of 4 patients with ratHEV-specific IgG antibodies were not detected by a commercial HEV IgG antibody ELISA kit. For this reason, for the evaluation ratHEV IgG antibodies of large populations, the use of a specific ELISA for ratHEV or the use of a mix antigen ELISA is needed. Several in-house ELISAs have been evaluated and demonstrated to be useful for the detection of ratHEV-specific IgG antibodies in different patients’ subsets [24–26]. Nevertheless, because of the limited number of cases diagnosed, their clinical validation is challenging. Recently, a parallel IgG enzyme immunoassay (EIA) system that can simultaneously detect anti-HEV antibodies and differentiate between individuals with HEV exposure and HEV-C1 (ratHEV) exposure, has been validated for the determination of ratHEV IgG seroprevalence [25]. Despite its validation, only samples from 16 ratHEV patients were included, this set comprises 80% of all patients with ratHEV reported worldwide. This assay has also been shown to be useful for the determination of the ratHEV seroprevalence in animal populations [33], reporting a 1.2% and 1.5% in dogs and cats, respectively. Consequently, this ELISA represents the current most promising tool for assessing the prevalence of ratHEV infection in large populations.

In conclusion, our study underscores the need for enhanced surveillance and targeted preventive measures for ratHEV. Given the significant exposure rates and potential public health impact, ratHEV should be considered a public health concern. Targeted studies with proper control groups are necessary to evaluate high-risk populations and transmission routes more accurately. For that, the development of specific and reliable serological tools is essential for addressing this emerging public health threat.

Acknowledgements

We gratefully acknowledge Laura Ruiz Torres and Ismael Zafra Soto for their technical support in sample processing and analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the data generated or analyzed during the study are included in the article. The datasets used and/or analyzed during the present research project are available from the corresponding author upon reasonable request. The viral sequence is available in GenBank under accession number PP256210.

Authors’ contributions

MCJ, ARJ and AR were involved in the study design and conception, interpretation of the data, drafting of the manuscript, study supervision, and funding obtention. LML, MGC and DCA recruited patients. DCM, AC, MG, IPV, and ABPJ were involved in clinical and microbiological evaluation, obtained informed consent and samples storage. MCJ, PLL, JCG and LRM performed RNA extraction and molecular determinations, phylogenetic analysis and GenBank submission. MCJ and TGG were involved in DB analyses with the advice and supervision of RGU. All authors have revised the manuscript and approved its publication.
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References

1 Wang B, Harms D, Yang X-L, et al. Orthohepevirus C: an expanding species of emerging hepatitis E virus variants. Pathogens. 2020;9 :154. doi:10.3390/pathogens9030154 32106525
2 Sridhar S, Yip CCY, Lo KHY, et al. Hepatitis E virus species C infection in humans, Hong Kong. Clin Infect Dis. 2022;75 :288–296. doi:10.1093/cid/ciab919 34718428
3 Sridhar S, Yip CCY, Wu S, et al. Rat hepatitis E virus as cause of persistent hepatitis after liver transplant. Emerg Infect Dis. 2018;24 :2241–2250. doi:10.3201/eid2412.180937 30457530
4 Andonov A, Robbins M, Borlang J, et al. Rat hepatitis E virus linked to severe acute hepatitis in an immunocompetent patient. J Infect Dis. 2019;220 (6 ):951–955. doi:10.1093/infdis/jiz025 30649379
5 Rivero-Juarez A, Frias M, Perez AB, et al. Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: first report in Europe. J Hepatol. 2022;77 (2 ):326–331. doi:10.1016/j.jhep.2022.01.028 35167911
6 Rodriguez C, Marchand S, Sessa A, et al. Orthohepevirus C hepatitis, an underdiagnosed disease? [published online ahead of print]. J Hepatol. 2023 Feb 18;S0168-8278 (23 ):00096–X. doi:10.1016/j.jhep.2023.02.00
7 Caballero-Gómez J, Rivero-Juarez A, Jurado-Tarifa E, et al. Serological and molecular survey of hepatitis E virus in cats and dogs in Spain. Transbound Emerg Dis. 2022;69 :240–248. doi:10.1111/tbed.14437 34951935
8 Rios-Muñoz L, Gonzálvez M, Caballero-Gomez J, et al. Detection of rat hepatitis E virus in pigs: Spain. Emerg Infect Dis. 2023. 2024;30 (4 ):823–826. doi:10.3201/eid3004.231629
9 Reuter G, Boros Á, Pankovics P. Review of hepatitis E virus in rats: evident risk of species orthohepevirus C to human zoonotic infection and disease. Viruses. 2020;12 :1148. doi:10.3390/v12101148 33050353
10 Sridhar S, Chew NF, Situ J, et al. Risk of hepatitis E among persons who inject drugs in Hong Kong: a qualitative and quantitative serological analysis. Microorganisms. 2020;8 (5 ):675. doi:10.3390/microorganisms8050675 32384808
11 Corona-Mata D, Rivero-Juárez A, Camacho Á, et al. Efficacy of a comprehensive strategy for the detection and treatment of hepatitis C infection in a population attending addiction centers. Front Public Health. 2023;11 :1092960. doi:10.3389/fpubh.2023.1092960 36817894
12 Casares-Jimenez M, Rivero-Juarez A, Lopez-Lopez P, et al. Rat hepatitis E virus (Rocahepevirus ratti) in people living with HIV. Emerg Microbes Infect. 2024;13 (1 ):2295389. doi:10.1080/22221751.2023.2295389 38095070
13 Dremsek P, Wenzel JJ, Johne R, et al. Seroprevalence study in forestry workers from eastern Germany using novel genotype 3- and rat hepatitis E virus-specific immunoglobulin G ELISAs. Med Microbiol Immunol. 2012;201 (2 ):189–200. doi:10.1007/s00430-011-0221-2 22179131
14 Lundkvist A, Meisel H, Koletzki D, et al. Mapping of B-cell epitopes in the nucleocapsid protein of puumala hantavirus. Viral Immunol. 2002;15 (1 ):177–192. doi:10.1089/088282402317340323 11952140
15 Chen C-S, Sullivan S, Anderson T, et al. Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip. Mol Cell Proteomics. 2009;8 :1765–1776. doi:10.1074/mcp.m800593-mcp200 19357087
16 Yip CW, Hon CC, Zeng F, et al. Naturally occurring anti-Escherichia coli protein antibodies in the sera of healthy humans cause analytical interference in a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for serodiagnosis of severe acute respiratory syndrome. Clin Vaccine Immunol: CVI. 2007;14 (1 ):99–101. doi:10.1128/CVI.00136-06 17108287
17 Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnol Adv 2012;30 (Issue 5 ):1102–1107. doi:10.1016/j.biotechadv.2011.09.013 21968145
18 Kubickova B, Schenk JA, Ramm F, et al. A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen. Appl Microbiol Biotechnol 2021;105 :4957–4973. doi:10.1007/s00253-021-11342-7 34129082
19 Rivero-Juarez A, Frias M, Lopez-Lopez P, et al. Hepatitis E 3ra genotype infection in people living with HIV in Spain. Front Microbiol. 2020;11 :564486. doi:10.3389/fmicb.2020.564486. Published 2020 Sep 11.33716992
20 Parraud D, Lhomme S, Péron JM, et al. Rat hepatitis E virus: presence in humans in south-western France? Front Med (Lausanne). 2021;8 :726363.34540871
21 Mulyanto, Suparyatmo JB, Andayani IG, et al. Marked genomic heterogeneity of rat hepatitis E virus strains in Indonesia demonstrated on a full-length genome analysis. Virus Res. 2014;179 :102–112. doi:10.1016/j.virusres.2013.10.029 24231359
22 Casares-Jimenez M, Garcia-Garcia T, Suárez-Cárdenas JM, et al. Correlation of hepatitis E and rat hepatitis E viruses urban wastewater monitoring and clinical cases. Sci Total Environ 2024;908 :168203. doi:10.1016/j.scitotenv.2023.168203 37914110
23 Haring VC, Litz B, Jacob J, et al. Small in size but huge as reservoir – insights into the virome of European white-toothed shrews. bioRxiv. 2023. doi:10.1101/2023.11.14.567014
24 Shimizu K, Hamaguchi S, Ngo CC, et al. Serological evidence of infection with rodent-borne hepatitis E virus HEV-C1 or antigenically related virus in humans. J Vet Med Sci. 2016;78 :1677–1681. doi:10.1292/jvms.16-0200 27499185
25 Situ J, Hon-Yin Lo K, Cai JP, et al. An immunoassay system to investigate epidemiology of rocahepevirus ratti (rat hepatitis E virus) infection in humans. JHEP Rep Innov Hepatol. 2023;5 (9 ):100793. doi:10.1016/j.jhepr.2023.100793
26 Dremsek P, Joel S, Baechlein C, et al. Hepatitis E virus seroprevalence of domestic pigs in Germany determined by a novel in-house and two reference ELISAs. J Virol Methods. 2013;190 (1-2 ):11–16. doi:10.1016/j.jviromet.2013.03.010 23523888
27 Lugoboni F, Pajusco B, Albiero A, et al. Hepatitis A virus among drug users and the role of vaccination: a review. Front Psychiatry. 2012 Jan 12;2 :79. doi:10.3389/fpsyt.2011.00079 22347865
28 Luquero FJ, Vallejo F, Fuente LdL, et al. The role of injection versus socioeconomic factors in hepatitis A virus infection among young heroin users: implications for vaccination policies. Vaccine. 2009;27 (20 ):2674–2679. doi:10.1016/j.vaccine.2009.02.056 19428878
29 Yrondi A, Salles J, Péron JM, et al. The prevalence of hepatitis E in a patient cohort presenting with addictive injection behavior. Front. Psychiatry. 2019;10 :832. doi:10.3389/fpsyt.2019.00832 31798477
30 Christensen PB, Engle RE, Jacobsen SEH, et al. High prevalence of hepatitis E antibodies among Danish prisoners and drug users. J Med Virol 2002;66 :49–55. doi:10.1002/jmv.2110 11748658
31 Castro VOL, Tejada-Strop A, Weis SMS, et al. Evidence of hepatitis E virus infections among persons who use crack cocaine from the Midwest region of Brazil. J Med Virol 2019;91 :151–154. doi:10.1002/jmv.25288 30133759
32 Sridhar S, Situ J, Cai JP, et al. Multimodal investigation of rat hepatitis E virus antigenicity: implications for infection, diagnostics, and vaccine efficacy. J Hepatol 2021;74 (6 ):1315–1324. doi:10.1016/j.jhep.2020.12.028 33845058
33 Shun EH, Situ J, Tsoi JY, et al. Rat hepatitis E virus (Rocahepevirus ratti) exposure in cats and dogs, Hong Kong. Emerg Microbes Infect. 2024;13 (1 ):2337671. doi:10.1080/22221751.2024.2337671 38551320
