Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: First report in Europe

Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: First report in Europe Graphical abstract Cohort 1 Acute hepatitis of unknown aethiology N = 169 Cohort 2 Acute hepatitis E N = 98 Orthohepevirus C RNA testing (NCT05062967) IgM-/RNA+ n = 13 IgM+/RNA- n = 40 IgM+/RNA+ n = 45 0 case 0 case 1 case 2.5% (95% CI: 0.06-13.1) Patient 1 (Genbank OK082152) Severe acute hepatitis and deceased Severe acute hepatitis and deceased Patient 2 (Genbank OK082153) Patient 3 (Genbank OK082154) Self-limited acute hepatitis Self-limited acute hepatitis 2 cases 1.18% (95% CI: 0.2-3.8) Highlights First cases of acute hepatitis related to Orthohepevirus C infection in Europe. Second registered death related to Orthohepevirus C infection worldwide in an immunosuppressed individual. Screening for Orthohepevirus C RNA should be evaluated in all patients with acute hepatitis. Authors Antonio Rivero-Juarez, Mario Frias, Ana Belen Perez, ., Encarnación Ramirez-Arellano, Juan Carlos Alados, Antonio Rivero Correspondence [email protected] (A. Rivero-Juarez). Lay summary We describe the first cases of acute hepatitis related to rat hepatitis E virus in Europe. The prevalence found in our study suggest that rat hepatitisEviruscouldbeconsidered an emerging disease in Europe. https://doi.org/10.1016/j.jhep.2022.01.028 © 2022 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Hepatol. 2022, 77, 326–331 Research Article Viral Hepatitis Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: First report in Europe Antonio Rivero-Juarez 1,2, * ,† , Mario Frias 1,2,† , Ana Belen Perez 2,3 , Juan Antonio Pineda 2,4 , Gabriel Reina 5 , Ana Fuentes-Lopez 2,6,7 , Carolina Freyre-Carrillo 8 , Encarnación Ramirez-Arellano 9 , Juan Carlos Alados 10 , Antonio Rivero 1,2 , For the HEPAVIR and GEHEP-014 Study Groups 1 Unit of Infectious Diseases, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Universidad de Córdoba (UCO), Córdoba, Spain; 2 CIBERINFEC, ISCIII - CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain; 3 Clinical Microbiology Unit, Hospital Universitario Reina Sofía, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain; 4 Unit of Infectious Diseases and Microbiology, Hospital Universitario de Valme, Seville, Spain; 5 Microbiology Department, Clínica Universidad de Navarra, STUN, Institute of Tropical Health, Universidad de Navarra, diSNA, Navarra Institute for Health Research, Pamplona, Spain; 6 Clinical Microbiology Unit, Hospital Universitario Clínico San Cecilio, Granada, Spain; 7 Instituto de Investigación Biosanitaria Ibs.Granada, Granada, Spain; 8 Clinical Microbiology Unit, University Hospital of Puerto Real, Cádiz, Spain; 9 Infectious Diseases, Microbiology and Preventive Medicine Unit, Virgen Macarena Univ. Hospital, and Department of Medicine, University of Sevilla / Biomedicine Institute of Sevilla, Sevilla, Spain; 10 Clinical Microbiology Unit, Hospital Universitario de Jerez, Cádiz, Spain Background & Aim: Hepatitis E virus (HEV) was considered the only member of the Hepeviridae family with zoonotic potential. Nevertheless, this consideration has been reassessed owing to several reported cases of acute and chronic hepatitis linked to the Orthohepevirus C genus. Because the circulation of Orthohepevirus C in rodents has been described worldwide, the risk of zoonotic transmission is plausibly global. Methods: Orthohepevirus C RNA was retrospectively evaluated in 2 cohorts of patients in Spain. The first cohort included patients with acute hepatitis without etiological diagnosis after screening for hepatotropic virus infection. The second cohort included patients diagnosed with acute HEV infection, defined as positivity for anti-HEV-IgM antibodies and/or detectable HEV RNA in serum. Results: Cohort 1 comprised 169 patients (64.4% male, median age 43 years) and cohort 2 comprised 98 individuals (68.3% male, median age 45 years). Of the individuals included in Cohort 1, two (1.18%; 95% CI 0.2-3.8) had detectable Orthohepevirus C RNA in serum. In Cohort 2, of the 98 included patients, 58 showed detectable HEV RNA, while 40 only showed positivity for IgM antibodies. Among those bearing only IgM antibodies, Orthohepevirus C RNA was detected in 1 (2.5%; 95% CI 0.06-13.1) individual. All strains were consistent with genotype C1. The infection resulted in mild self-limiting acute hepatitis in 2 patients. Infection caused severe acute hepatitis in the remaining patient who died as a result of liver and renal failure. Conclusions: We described 3 cases of Orthohepevirus C in patients with acute hepatitis, resulting in the first description of this infection in Europe. The prevalence obtained in our study suggests that Orthohepevirus C could be an emerging disease in Europe. Lay summary: We describe the first cases of acute hepatitis related to rat hepatitis E virus in Europe. The prevalence found in our study suggest that rat hepatitis E virus could be considered an emerging disease in Europe. © 2022 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). Introduction The Orthohepevirus genus comprises single-strain RNA viruses including 4 species: Orthohepevirus A, B, C and D. 1 Orthohepevirus A, known as hepatitis E virus (HEV), is one of the major causes of acute hepatitis worldwide. 2,3 According to genome sequences, HEVs present 8 major genotypes, 4 whose circulation can be limited to humans through consumption of fecal-contaminated water in Asian and African countries (genotypes 1 and 2) or present a worldwide distribution circulating among humans and a wide number of mammalian species with zoonotic transmission through the consumption of raw or undercooked meat (genotypes 3 to 8). 5,6 Therefore, HEV is a zoonotic disease that is considered a major public health issue in both high- and lowincome countries. 7,8 In contrast, the other 3 Orthohepevirus species seemed to lack zoonotic threat, and their circulation seemed to be limited to their main host: Orthohepevirus Bin birds, 9 Orthohepevirus C in mustelids and rodents, 10 and Orthohepevirus D in bats. 11 Nevertheless, this consideration was reassessed because of recent studies. In 2018, a case of rat HEV infection in a liver transplant recipient was reported in Hong Kong. 12 Thereafter, in a large screening population in the same setting, 7 additional cases of Orthohepevirus C infection were Keywords: Hepatitis E; Orthohepevirus C; acute hepatitis; rodent; zoonosis. Received 6 November 2021; received in revised form 22 January 2022; accepted 31 January 2022; available online 12 February 2022 *Corresponding author. Address: Virología Clínica y Zoonosis, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Avenida Menedez Pidal, s/n. 14004, Córdoba, Spain; Tel.: +34 957 21 38 06. E-mail address: [email protected] (A. Rivero-Juarez). † These authors contributed equally to this work. https://doi.org/10.1016/j.jhep.2022.01.028 Journal of Hepatology 2022 vol. 77 j326–331 Research Article Viral Hepatitis described. 13 Interestingly, in this study, the author demonstrated epizootic transmission because the virus was identified in rodents sampled from the same district. Despite most patients reported in these studies present underlying chronic conditions, most of them with immunosuppression, several cases were immunocompetent. 13 After that, a prospective screening program for Orthohepevirus C in Hong Kong has been set up, demonstrating that this virus is an emerging cause of acute hepatitis in this setting, 14 with the number of known cases rising to 16. Because the circulation of Orthohepevirus C in rodents has been described worldwide (supplementary information), the risk of zoonotic transmission is plausible globally and not restricted to a specific area. Notably, 1 case outside Hong Kong has been reported, and it showed severe acute hepatitis. This was an immunocompetent patient who presumably acquired the infection in central Africa. 15 This finding justifies the active search for Orthohepevirus C cases in other countries. A recent study conducted in France retrospectively analyzed samples from 224 individuals for Orthohepevirus CRNA. 16 They did not find any case, nevertheless most individuals included were immunocompromised and only the 63% had abnormal liver function tests. 16 Therefore, the aim of our study was to evaluate the prevalence of Orthohepevirus C infection in immunocompetent individuals with acute hepatitis from a country where Orthohepevirus C has been identified in rodents, 17 and to elucidate the role of this virus as an etiological agent of acute hepatitis. Patients and methods Patients and setting The study population included 267 patients with acute hepatitis in follow-up at 7 reference hospitals in Spain and belonged to 2 different cohorts (ClinicalTrials.gov Identifier: NCT05062967). The study period encompassed 1 January 2018 (both cohort setups) to 1st September 2021 (study censored date). The first cohort comprises 169 patients. The criteria for inclusion in this cohort were i) clinical and biological manifestations compatible with acute hepatitis, ii) alanine aminotransferase level 3x the upper limit of normal, and iii) no etiological diagnosis after screening for hepatotropic virus infection. This screening included serological and/or molecular markers for hepatitis A virus (IgM antibodies), hepatitis B virus (HBsAg, HBcAc, and viral DNA), hepatitis C virus (IgG antibodies and viral RNA), HEV (IgM antibodies and viral RNA), cytomegalovirus (IgM antibodies), and Epstein-Barr virus (IgM antibodies). The second cohort included 98 patients diagnosed with acute HEV infection. Acute HEV infection was defined as i) clinical and biological manifestation compatible with acute hepatitis and ii) positivity for anti-HEV-IgM antibodies and/or detectable HEV RNA in serum. Serological and molecular screening for HEV Information about all reagents, software and biological samples employed in the study can be found in the supplementary CTAT table. HEV molecular and serological markers for HEV infection were centrally evaluated at the Clinical Virology and Zoonoses laboratory of the Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC). Anti-HEV antibodies were evaluated by enzyme immunoassay using the HEV-IgM kit and HEV- IgG kit developed by Wantai (Beijing Wantai Biological Pharmacy Enterprise Ltd., Beijing, China) by an automated procedure using DYNEX DS2 ® (DYNEX Technologies. Sullyfield Circle Chantilly, VA, USA). For HEV RNA evaluation, we applied a protocol developed and validated by our group for Orthohepevirus A detection, with a detection limit set at 21 IU/ml. 18 Briefly, RNA was extracted from 400 l l of serum using the QIAamp Mini Elute virus spin kit (Qiagen, Hilden, Germany) and QIAcube (Qiagen, Hilden, Germany). The purified RNA was eluted in a 50 l l volume using 25 l l for reverse-transcription quantitative PCR using the One-Step PCR Kit (Qiagen, Hilden, Germany). The sensitivity of the ELISA for rat HEV-derived IgG and IgM employed at screening has been estimated to be 70% and 40%, respectively. 19 Molecular evaluation for Orthohepevirus C infection All patients were retrospectively evaluated for Orthohepevirus C infection using broad-spectrum nested PCR targeting the RdRp gene, as described previously. 20 Ten microliters of nucleic acid extraction were used in combination with the One-Step reversetranscription quantitative PCR kit (Qiagen, Hilden, Germany) for the first round of PCR and 5 l l of the first PCR product using Promega master Mix (Madison, USA) for the second reaction. We used the 1st World Health Organization International Standard for HEV RNA nucleic acid amplification test-based assays – consistent with HEV genotype 3a and provided by the Paul- Ehrlich-Institut (PEI code 6219/10) –as a positive control. The lyophilizate material was reconstituted with 500 l l of diethyl pyrocarbonate-treated water (Thermo Fisher Scientific, Waltham, MA, USA) using 200 l l for nucleic acid extraction employing the same procedure used for serum samples. The amplicons were examined on 1.5% agarose gels stained with RedSafe TM Nucleic Acid Staining solution. PCR products with the correct target size (approximately 330 nucleotides) were purified using Illustra TM ExoProStar TM . 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). Statistical analysis Categorical variables were expressed as the number of cases (percentage), and continuous variables were expressed as the median (IQR). The prevalence of Orthohepevirus C infection was calculated in each cohort. For prevalence, the 2-sided 95% CI was calculated using the exact binomial distribution. The Orthohepevirus species were assigned using the HEVnet genotyping tool (https://www.rivm.nl/mpf/typingtool/hev/) and confirmed by BLAST. 21 Sequence alignments were generated by the MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Phylogenetic trees were constructed using the maximum likelihood method using 68 Orthohepevirus C strains. Information about these strains can be found in Table S1. Four sequences belonging to Orthohepevirus A (M73218), Orthohepevirus B (GU954430), Orthohepevirus D (KX513953), and the proposed Orthohepevirus E (KF951328) were included as outgroups to root the tree. Two sequences previously isolated in 2 Rattus rattus specimens sampled in 2013 in South Spain were also included (KY938026 and KY938027). 17 The final tree was obtained with MEGA Software (Version 7) using the bootstrap method (bootstrapped with 1,000 replicates). Ethics statement This study was designed and conducted in accordance with the Declaration of Helsinki. The Ethics and Clinical Trials Committee Journal of Hepatology 2022 vol. 77 j326–331 327 (CEIC) of Córdoba approved the study protocol (protocol reference number 5081), and informed consent was obtained from each patient. The Sistema Sanitario Público Andaluz (SSPA) Biobank coordinated the collection, processing, handling and assignment of the biological samples used in this study in accordance with the standard procedures established for this purpose. Results Population Of the 169 patients included in Cohort 1, 109 (64.4%) were male, with a median (IQR) age of 43 years (36-55). This population showed a median (IQR) level of 157 U/L (101-601) for alanine aminotransferase, 95 U/L (23-366) for aspartate aminotransferase, 114 U/L (40-272) for gamma-glutamyltransferase, and 0.7 mg/dl (0.5-4) for bilirubin. Among the 98 who constituted Cohort 2, 67 (68.3%) were male, and the median (IQR) age was 45 (34-53) years. The median alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, and bilirubin levels in this cohort were 131 U/L (37-435), 99 U/L (27-394), 128 U/L (41-289), and 0.7 mg/dl (0.6-4.4), respectively. Forty-five individuals (45.9%) were positive for both anti-HEV-IgM antibodies and HEV RNA, 40 (40.9%) exhibited positivity only for IgM antibodies, and 13 (13.2%) showed detectable HEV RNA but were negative for IgM antibodies. Globally, 85 (86.7%) patients were positive for IgM anti-HEV, and 58 (59.1%) showed detectable viral RNA in serum. The viral genotype was assigned in all individuals with detectable HEV RNA: 51 (86.4%) cases were genotype 3f, 3 (5.2%) were genotype 3 m, 1 (3.2%) was genotype 3e, and subtype could not be defined in 3 (5.2%) patients bearing genotype 3 infection. Prevalence of Orthohepevirus C infection The screening results for Orthohepevirus C are shown in Fig. 1.Of the individuals included in Cohort 1, 2 (1.18%; 95% CI 0.2-3.8) had detectable Orthohepevirus C RNA in the serum. In Cohort 2, of the 98 included patients, 58 had detectable HEV RNA, while 40 only showed positivity for IgM antibodies. Orthohepevirus C was not detected in any of the patients with detectable HEV RNA. Instead, Orthohepevirus C RNA was detected in 1 (2.5%; 95% CI 0.06-13.1) individual among those bearing only IgM antibodies. The partial genome of the RdRp gene showed that the 3 strains were consistent with genotype C1 (Fig. 2). These sequences are available at GenBank under accession numbers OK082152, OK082153, and OK082154. By BLAST, we found high homology (99.1%-98.5%) with Orthohepevirus C strains isolated in Rattus rattus (MH400712, MH400713 and MH400717) and Rattus norvegicus (MH400714 to MH400716) specimens from Lithuania sampled in 2016, 22 and with the sequence GU345043, originating from a Rattus norvegicus in Germany in 2009. Additionally, the sequences identified in our study were closely related to Orthohepevirus C strains previously isolated in 2 Rattus rattus specimens from southern Spain (KY938026 and KY938027) (Fig. 2). 17 Cases of Orthohepevirus C In Table 1, we summarized the main clinical and epidemiological features of the 3 cases of Orthohepevirus C infection. All patients were male, Patient 1 and Patient 2 were inhabitants of southern Spain, and Patient 3 lived in northern Spain. None of them reported contact with animals (including pets, farm animals, wild animals, or hunting), conscious contact with rodents, or travel outside Spain in the previous months. As risk factors for HEV infection, Patient 1 and Patient 2 reported consumption of undercooked pork in the 2 weeks prior to diagnosis. Patient 3 reported being a cleaner. Orthohepevirus C infection in Patient 2 and Patient 3 resulted in mild acute hepatitis with selfresolution. Meanwhile, Patient 1 was admitted to the hospital because of severe acute hepatitis and died 9 days after admission because of liver and renal failure. Discussion We report the first cases of Orthohepevirus C infection in Europe. Orthohepevirus C was identified as the etiological agent of acute Populations tested (NCT05062967) Cohort 1 N = 169 Orthohepevirus C+ n = 2 1.18% (95% CI 0.2-3.8) HEV IgM- and HEV RNA+ n = 13 Orthohepevirus C+ n = 0 Cohort 2 N = 98 HEV IgM+ and HEV RNA- n = 40 Orthohepevirus C+ n = 1 2.5% (95% CI 0.06-13.1 ) HEV IgM and HEV RNA+ n = 45 Orthohepevirus C+ n = 0 Fig. 1. Study diagram flow and screening results for Orthohepevirus C. HEV, hepatitis E virus. 328 Journal of Hepatology 2022 vol. 77 j326–331 Research Article Viral Hepatitis hepatitis in 2.5% of patients with a diagnosis of acute HEV based only on the presence of IgM antibodies (1 out 40 cases) and in 1.18% of individuals with acute hepatitis not related to hepatotropic virus infection (2 out 169). Our findings suggest that Orthohepevirus C could be an emerging infectious disease. In 2 cases, the infection course was symptomatic self-limiting hepatitis, while in the other case, the infection triggered fatal liver and renal failure. Our findings are in line with previous cases of Orthohepevirus C infection, showing that the clinical course of the infection ranges from mild and self-limiting acute hepatitis to severe fatal outcomes. 13,14 Although the first evidence of Orthohepevirus C infection was aimed at the context of underlying immunosuppression, 12,13 other cases were documented in an immunocompetent individual. 15 In this sense, 2 cases identified in our study did not suffer from immunosuppression, suggesting that this condition is not necessary to be susceptible to the infection and to present symptomatic infection. However, although the number of cases reported of Orthohepevirus C infection is low, the course of the infection appears to be worse in individuals with underlying GU345042_Rodent_Germany MH400712_Rodent_Lithuania MH400714_Rodent_Lithuania MH400715_Rodent_Lithuania MH400713_Rodent_Lithuania MH400717_Rodent_Lithuania OK082153_Patient_2 OK082154_Patient_3 MH400716_Rodent_Lithuania OK082152_Patient_1 MN660075_Cattle_Turkey MW795383_Rodent_Germany GQ504009_Rodent_Germany GU345043_Rodent_Germany 89 JF516246_Rodent_USA KM516906_Rodent_USA 99 JN167538_Rodent_Germany KX774671_Rodent_Germany 81 JN167537_Rodent_Germany KX774641_Rodent_Germany 93 AB847308_Rodent_Indonesia KY938026_Rodent_Spain KY938027_Rodent_Spain 77 MW020590_Wastewater_Sweden MW020591_Wastewater_Sweden 96 KX774669_Rodent_Switzerland KC294199_Rodent_Denmark KX774670_Rodent_Germany KX774667_Rodent_Denmark KX774672_Rodent_Germany MH729810_Rodent_China MH729811_Rodent_China KX844624_Rodent_China KX774668_Rodent_Austria MW149254_Rodent_Sweden KX774673_Rodent_France KY938025_Rodent_Belgium KY938011_Rodent_Greece KY938012_Rodent_Greece KX774664_Rodent_France KX774665_Rodent_France 96 MK050105_Human_Canada LC549186_Rodent_China LC549187_Human_China 99 MT085259_Rodent_Laos MT085260_Rodent_Thailand 86 LC088965_Rodent_Myanmar AB847306_Rodent_Indonesia AB847309_Rodent_Indonesia MT085261_Rodent_Camboia KY659377_Rodent_China KY659378_Rodent_China 89 KY659379_Rodent_China LC549184_Rodent_China 75 LC225389_Rodent_Indonesia MN450853_Human_Hong_Kong AB847305_Rodent_Indonesia LC145325_Rodent_Indonesia LC225388_Rodent_Indonesia 81 KC465990_Rodent_China LC549185_Rodent_China 98 AB847307_Rodent_Indonesia JX120573_Rodent_Vietnam MG813927_Rodent_Hong_Kong MN450851_Human_Hong_Kong 98 MN450852_Human_Hong_Kong MN450854_Human_Hong_Kong 96 82 JN998606_Ferret_Netherlands JN998607_Ferret_Netherlands 99 AB890374_Ferret_Japan AB898203_Ferret_Japan 92 AB898205_Ferret_Japan MN822837_Ferret_China MN822838_Ferret_China 76 AB898200_Ferret_Japan AB898202_Ferret_Japan AB898201_Ferret_Japan AB898204_Ferret_Japan AB898199_Ferret_Japan LC177789_Ferret_USA LC177790_Ferret_USA LC177791_Ferret_USA LC177788_Ferret_USA LC177792_Ferret_USA 75 97 88 98 MG020022_Rodent_China MG019963_Rodent_China MG020023_Rodent_China MG019966_Rodent_China MH581168_Rodent_Hungary 92 99 88 KU670940_Falco_Hungary KF951328_E M73218_A 88 GU954430_B KX513953_D 88 Fig. 2. The evolutionary history was inferred by using the maximum likelihood method based on the Jukes-Cantor model. The bootstrap consensus tree inferred from 1,000 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-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with superior log likelihood value. The analysis involved 94 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions with less than 95% site coverage were eliminated. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Light blue circle highlights the sequences identified in the present study. Light blue diamond highlights those sequences identified in Spanish Rattus rattus specimens. Journal of Hepatology 2022 vol. 77 j326–331 329 immunosuppression. In this sense, the only patient suffering from immunosuppression showed severe acute hepatitis with a rapid deterioration of liver function and death and is the second registered death related to Orthohepevirus C infection. 13 In this sense, the first registered death linked to Orthohepevirus C infection was recorded in a kidney transplant recipient. It should be noted that this patient also suffered from a severe underlying disease (metastatic cancer), which might have affected the clinical course of the hepatitis. Thus, close surveillance of Orthohepevirus C could be needed not only in patients with underlying immunosuppression, but also in patients with severe underlying comorbidities. Based on our current knowledge, from a clinical point of view, Orthohepevirus C and HEV infection seem to be indistinguishable, and cross-reactivity antibodies between Orthohepevirus C and HEV have been observed. 19 Therefore, 1 case in our study and 6 cases in the Hong Kong study exhibited HEV-IgM antibodies with undetectable HEV RNA. In the same way, the proportion of Orthohepevirus C-infected individuals among those carrying HEV-IgM antibodies was similar between our study (2.5%) and the study conducted in Hong Kong (2.9%). 13 Therefore, based on both studies, the evaluation of Orthohepevirus C RNA in all patients with HEV-IgM antibodies with undetectable HEV RNA should be recommended, independent of the origin. In contrast, in both studies, 8 individuals lacking HEV-IgM antibodies were identified. 13,14 These findings suggest that the presence of HEV- IgM antibodies has a low sensitivity for Orthohepevirus C diagnosis, and therefore, its use as a diagnosis-based algorithm for Orthohepevirus C might be very limited. 19 Thus, based on this, screening for Orthohepevirus C RNA in patients with acute hepatitis should be evaluated independently of HEV serological patterns. The main hosts of Orthohepevirus C are rodents, which have a global distribution and circulation. 23–25 For this reason, close contact with this species or with its droppings could be the main transmission route. 12 In our study, similar to the study conducted in Hong Kong, we found high homology between human viral sequences and those identified in rodents from the same area, suggesting that the route of infection is related to direct or indirect contact with rodents. 13,14 However, none of the cases described in our study and only 1 of the cases described in Hong Kong reported contact with rodents. 13 Thus, alternative transmission routes need to be evaluated. Several limitations should be noted. First, the HEV serological pattern of the patients included in the study was assessed using only 1 assay. Because of the variation in the sensitivity of the available HEV commercial assays for Orthohepevirus C-derived antibodies, the categorization of the population could change depending on the assays employed. Second, although patients were included prospectively in both cohorts, because of the retrospective character of the analysis, epidemiological investigations were limited. Consequently, it is possible to fail to detect other risk factors associated with Orthohepevirus C acquisition. Finally, a survey of Orthohepevirus C in rodents from the same setting was not performed, so we only included 2 viral sequences isolated in rodents from Spain obtained in a previous study. This point has an evident negative impact on the phylogenetic traceability of the cases. Prospective Orthohepevirus C surveys in both rat and human populations from our setting are warranted. In conclusion, the prevalence found in our study suggests that Orthohepevirus C may be an emerging cause of acute hepatitis in Europe, which could be misdiagnosed because of cross-reactivity with HEV-derived antibodies and the lack of molecular screening. Table 1. Epidemiological and clinical characteristics of patients infected by rat HEV. Variable Patient 1 Patient 2 Patient 3 Cohort of origin Cohort 2 Cohort 1 Cohort 1 Age (years) 62 30 54 Sex Male Male Male Year of the sample 2020 2018 2019 Region Southern Spain Southern Spain Northern Spain Location Córdoba Seville Vitoria Significant underlying disorder Metastatic oral cancer No No Concomitant medication Amoxicillin and clavulanic acid* dexamethasone † No Atorvastatine ‡ Exposure to domestic animals No No No Exposure to wild animals No No No Exposure to rodents No No No Consumption of raw or undercooked meat Yes Yes No Travel outside Spain No No No Blood transfusion No No No Other risk practice for HEV No Cleaning staff Unknown IgG anti-HEV antibodies Positive Negative Positive IgM anti-HEV antibodies Positive Negative Negative Peak ALT (U/L) 400 554 173 Peak AST (U/L) 340 549 137 Peak GGT (U/L) 630 72 536 Peak bilirubin (mg/dl) 8.4 0.7 1.09 Hospital admission Yes No No Intensive care unit admission Yes No No Infection outcome Death by liver and renal failure Self-limited Self-limited ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase; HEV, hepatitis E virus. *At a dose of 500 mg/125 mg every 8 hours. † At a dose of 4 mg twice daily. ‡ At a dose of 80 mg daily. 330 Journal of Hepatology 2022 vol. 77 j326–331 Research Article Viral Hepatitis Abbreviations HEV, hepatitis E virus. Financial support This work was supported by the Ministerio de Sanidad (RD12/ 0017/0012) integrated in the Plan Nacional de I+D+I and cofinanced by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER), Fundación para la Investigación en Salud (FIS) del Instituto Carlos III (PI19/00864 and PI21/00793). This research was supported by CIBER -Con- sorcio Centro de Investigación Biomédica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea –NextGenerationEU. ARJ is the recipient of a Miguel Servet Research Contract awarded by the Ministerio de Ciencia, Promoción y Universidades of Spain (CP18/00111). MF is the recipient of a Sara Borrell contract awarded by the Ministerio de Ciencia, Promoción y Universidades of Spain (CD18/00091). JAP has received a research extension grant from the Programa de Intensificación de la Actividad de Investigación del Servicio Nacional de Salud Carlos III (I3SNS). AR is the beneficiary of contracts for the intensification of research activity in the public health system awarded by the Ministerio de Ciencia, Promoción y Universidades of Spain (INT20-00028). The funders did not play any role in the design, conclusions, or interpretation of the study. Conflicts of interest The authors declare that there are no competing interests. Neither the authors nor their institutions have at any time received payment or services from a third party for any aspect of the submitted work (data monitoring board, study design, manuscript preparation, statistical analysis, and so on). Please refer to the accompanying ICMJE disclosure forms for further details. Authors’contributions ARJ was involved in the study design and conception, interpretation of the data, drafting of manuscript and study supervision. MF was involved in the serological and molecular determination. ABP, JAP, GR, JCA, AF, ERA and CF and were involved in the data acquisition and critical review of the manuscript. AR was involved in the study design and conception, interpretation of the data and drafting of manuscript. Data availability statement All data generated or analyzed during the study are included in this published article. The datasets used and/or analyzed during the present research project are available from the corresponding author upon reasonable request. Sequences are available in GenBank under accession numbers OK082152, OK082153, and OK082154. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jhep.2022.01.028. References Author names in bold designate shared co-first authorship. [1] Smith DB, Simmonds P. Members Of The International Committee On The Taxonomy Of Viruses Hepeviridae Study Group, Jameel S, Emerson SU, Harrison TJ, et al. Consensus proposals for classification of the family Hepeviridae. [published correction appears in J Gen Virol. 2015; 96(Pt 5): 1191-2]. J Gen Virol 2014;95(Pt 10):2223–2232. [2] Nimgaonkar I, Ding Q, Schwartz RE, Ploss A. Hepatitis E virus: advances and challenges. Nat Rev Gastroenterol Hepatol 2018;15:96–110. [3] World Health Organization. Global hepatitis report. 2017. Available at: http://www.who.int/hepatitis/publications/global-hepatitis-report2 017/en/. [4] Smith DB, Izopet J, Nicot F, Simmonds P, Jameel S, Meng XJ, et al. Update: proposed reference sequences for subtypes of hepatitis E virus (species Orthohepevirus A). J Gen Virol 2020;101(7):692–698. [5] Khuroo MS, Khuroo MS, Khuroo NS. Transmission of hepatitis E virus in developing countries. Viruses 2016;8(9):253. [6] Velavan TP, Pallerla SR, Johne R, Todt D, Steinmann E, Schemmerer M, et al. Hepatitis E: an update on One Health and clinical medicine. Liver Int 2021;41(7):1462–1473. [7] Kirkwood CD, Dobscha KR, Steele AD. Hepatitis E should be a global public health priority: recommendations for improving surveillance and prevention. Expert Rev Vaccin 2020;19(12):1129–1140. [8] Kupferschmidt K. Europe’s new hepatitis problem. Science 2016;353 (6302):862–863. [9] Sun P, Lin S, He S, Zhou EM, Zhao Q. Avian hepatitis E virus: with the trend of genotypes and host expansion. Front Microbiol 2019;10:1696. [10] 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(10):1148. [11] Johne R, Dremsek P, Reetz J, Heckel G, Hess M, Ulrich RG. Hepeviridae: an expanding family of vertebrate viruses. Infect Genet Evol 2014;27:212–229. [12] Sridhar S, Yip CCY, Wu S, Cai J, Zhang AJ, Leung KH, et al. Rat hepatitis E virus as cause of persistent hepatitis after liver transplant. Emerg Infect Dis 2018;24(12):2241–2250. [13] Sridhar S, Yip CC, Wu S, Chew NF, Leung KH, Chan JF, et al. Transmission of rat hepatitis E virus infection to humans in Hong Kong: a clinical and epidemiological analysis. Hepatology 2021;73(1):10–22. [14] Sridhar S, Yip CC, Lo KH, Wu S, Situ J, Chew NF, et al. Hepatitis E virus species C infection in humans, Hong Kong. Clin Infect Dis 2021. Epub ahead of print https://dx.doi.org/10.1093/cid/ciab919. [15] Andonov A, Robbins M, Borlang J, Cao J, Hatchette T, Stueck A, et al. Rat hepatitis E virus linked to severe acute hepatitis in an immunocompetent patient. J Infect Dis 2019;220(6):951–955. [16] Parraud D, Lhomme S, Péron JM, Da Silva I, Tavitian S, Kamar N, et al. Rat hepatitis E virus: presence in humans in South-Western France? Front Med 2021;8:726363. [17] Ryll R, Bernstein S, Heuser E, Schlegel M, Dremsek P, Zumpe M, et al. Detection of rat hepatitis E virus in wild Norway rats (Rattus norvegicus) and Black rats (Rattus rattus) from 11 European countries. Vet Microbiol 2017;208:58–68. [18] Frías M,López-López P, Zafra I, Caballero-Gómez J, Machuca I, Camacho Á, et al. Development and clinical validation of a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (Orthohepevirus A genus). J Clin Microbiol 2021;59(2). e02075-20. [19] Sridhar S, Situ J, Cai JP, Yip CC, Wu S, Zhang AJ, et al. Multimodal investigation of rat hepatitis E virus antigenicity: implications for infection, diagnostics, and vaccine efficacy. J Hepatol 2021;74(6):1315–1324. [20] Johne R, Plenge-Bönig A, Hess M, Ulrich RG, Reetz J, Schielke A. Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broadspectrum RT-PCR. J Gen Virol 2010;91(Pt 3):750–758. [21] Mulder AC, Kroneman A, Franz E, Vennema H, Tulen AD, Takkinen J, et al. HEVnet: a One Health, collaborative, interdisciplinary network and sequence data repository for enhanced hepatitis E virus molecular typing, characterisation and epidemiological investigations. Euro Surveill 2019;24(10):1800407. [22] Simanavicius M, Juskaite K, Verbickaite A, Jasiulionis M, Tamosiunas PL, Petraityte-Burneikiene R, et al. Detection of rat hepatitis E virus, but not human pathogenic hepatitis E virus genotype 1-4 infections in wild rats from Lithuania. Vet Microbiol 2018;221:129–133. [23] Murphy EG, Williams NJ, Jennings D, Chantrey J, Verin R, Grierson S, et al. First detection of hepatitis E virus (Orthohepevirus C) in wild brown rats (Rattus norvegicus) from Great Britain. Zoonoses Public Health 2019;66(6):686–694. [24] Li W, Guan D, Su J, Takeda N, Wakita T, Li TC, et al. High prevalence of rat hepatitis E virus inwild rats inChina. Vet Microbiol 2013;165(3-4):275–280. [25] Purcell RH, Engle RE, Rood MP, Kabrane-Lazizi Y, Nguyen HT, Govindarajan S, et al. Hepatitis E virus in rats, Los Angeles, California, USA. Emerg Infect Dis 2011;17(12):2216–2222. Journal of Hepatology 2022 vol. 77 j326–331 331