Investigating animal reservoirs for hepatitis E virus in Bangui, Central African Republic.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of enterotropic viral hepatitis, a major public health problem in many developing countries. In Central African Republic (CAR), HEV genotypes 1, 2, and 3 have been found to have an impact on human health. However, data on HEV in animal reservoirs are still lacking for CAR. Here, we investigated the presence of HEV in farmed pigs and goats in Bangui, the capital city of CAR, using molecular methods. METHODOLOGY: In a prospective study, fecal samples from 61 pigs and 39 goats from farms in five districts (2nd, 4th, 6th, 7th, 8th) of Bangui were collected and tested for HEV RNA by real-time RT-PCR. The samples were further analyzed by nested-PCR and sequenced to determine the genotype and subtype to which the virus belong. RESULTS: In total, 22/100 (22.0%) feces samples were successfully amplified for HEV RNA by real time RT-PCR. All positive samples were from pigs (22/61; 36.1%), while all goat samples were negative (0/39). Twelve HEV RNA samples (12/22 or 54.5%) were successfully amplified by nested RT-PCR, and subsequently sequenced. Phylogenetic analysis revealed that the obtained sequences clustered with subtype 3h and were genetically related to the human HEV sequences from CAR. CONCLUSION: This study confirms that pigs constitute an HEV reservoir, with genotype 3 being the major circulating strain. Further studies are needed to investigate other local reservoirs and to improve knowledge of the molecular epidemiology of HEV in CAR.

The molecular epidemiology of hepatitis E virus genotype 3 in Canada.

Autochthonous hepatitis E virus (HEV) infection is increasingly reported in industrialized countries and is mostly associated with zoonotic HEV genotype 3 (HEV-3). In this study, we examined the molecular epidemiology of 63 human clinical HEV-3 isolates in Canada between 2014 and 2022. Fifty-five samples were IgM positive, 45 samples were IgG positive and 44 were IgM and IgG positive. The majority of the isolates belong to the subtypes 3a, 3b, and 3j, with high sequence homology to Canadian swine and pork isolates. There were a few isolates that clustered with subtypes 3c, 3e, 3f, 3h, and 3g, and an isolate from chronic infection with a rabbit strain (3ra). Previous studies have demonstrated that the isolates from pork products and swine from Canada belong to subtypes 3a and 3b, therefore, domestic swine HEV is likely responsible for the majority of clinical HEV cases in Canada and further support the hypothesis that swine serve as the main reservoirs for HEV-3 infections. Understanding the associated risk of zoonotic HEV infection requires the establishment of sustainable surveillance strategies at the interface between humans, animals, and the environment within a One-Health framework.

Genomic characterization of Rocahepevirus ratti hepatitis E virus genotype C1 in Yunnan province of China.

The Rocahepevirus ratti hepatitis E virus genotype C1 (HEV-C1) has been documented to infect humans. However, the understanding of HEV-C1 remains constrained. This study aims to determine the prevalence and genomic characteristics of HEV-C1 in small animals in Yunnan province of southwestern China. A total of 444 liver tissues were collected from animals covering the orders Rodentia, Soricomorpha, Scandentia and Erinaceomorpha in three regions in Yunnan. Then Paslahepevirus balayani and Rocahepevirus were examined using RT-qPCR. The detection rate of Rocahepevirus was 12.95 % (36/278) in animals of order Rodentia, with 14.77 % (35/237) in Rattus tanezumi and 33.33 % (1/3) in Niviventer fulvescens. No Paslahepevirus balayani was detected. Additionally, two full-length Rocahepevirus sequences (MSE-17 and LHK-54) and thirty-three partial ORF1 sequences were amplified and determined to be HEV-C1. MSE-17 and LHK-54 shared moderate nucleotide identity (78.9 %-80.3 %) with HEV-C1 isolated in rats and humans. The HEV-C1 isolated from Niviventer fulvescens demonstrated a 100 % nucleotide identity with that from Rattus tanezumi. The rat HEV-C1 sequences isolated in our study and other Asian HEV-C1 sequences were phylogenetically distant from those isolated in North America and Europe. Furthermore, the two full-length sequences isolated in our study had less amino acid substitutions in the motifs of RNA-dependent RNA polymerase domain (F204L and L238F), compared with other Asian sequences. In summary, HEV-C1 commonly spreads in rats in Yunnan province of China. Our findings suggest a spatially associated phylogeny, and potential cross-species transmission of HEV-C1.

First detection and characterization of hepatitis E virus (Rocahepevirus ratti) from urban Norway rats (Rattus norvegicus) in the Republic of Korea.

Hepatitis E virus (HEV), an emerging zoonotic pathogen, poses a significant public health concern worldwide. Recently, rat HEV (Rocahepevirus ratti genotype C1; HEV-C1) has been reported to cause zoonotic infections and hepatitis in humans. Human infections with HEV-C1 are considered to be underestimated worldwide due to limited knowledge of transmission routes, genome epidemiology, and the risk assessment of zoonosis associated with these viruses. A total of 186 wild Norway rats (Rattus norvegicus) were collected from the Republic of Korea (ROK) between 2011 and 2021. The prevalence of HEV-C1 RNA was 8 of 180 (4.4%) by reverse-transcription polymerase chain reaction. We first reported three nearly whole-genome sequences of HEV-C1 newly acquired from urban rats in the ROK. Phylogenetic analysis demonstrated that Korea-indigenous HEV-C1 formed an independent genetic group with those derived from R. norvegicus rats in other countries, indicating geographical and genetic diversity. Our findings provide critical insights into the molecular prevalence, genome epidemiology, and zoonotic potential of Rocahepevirus. This report raises awareness of the presence of Rocahepevirus-related hepatitis E among physicians in the ROK.

No evidence of hepatitis E virus (HEV) infection among pet cats and dogs, and low seroprevalence of hepatitis E virus among pet rabbits in Poland.

The seroprevalence of Paslahepevirus balayani genotype 3 (hepatitis E virus genotype 3 - HEV-3; Hepeviridae family, genus Paslahepevirus) in pet cats, dogs and rabbits was evaluated. Samples from cats and dogs were collected from three veterinary practices from various parts of Poland: Poznan (wielkopolskie voivodeship), Przemysl (podkarpackie voivodeship) and Lublin (lubelskie voivodeship). Samples from rabbits were collected in Poznan. In total, serum samples from 90 cats, 82 dogs and 71 rabbits were selected and tested for specific anti-HEV-3 immunoglobulin (IgG) antibodies using a commercial ELISA test. Pathogen seroprevalence among rabbits was calculated at a 95% confidence interval (CI) for each gender, age (up to 12 months, 1-3 years, 4-7 years and over 8 years), symptoms group (healthy, gastrointestinal disorders, other disorders) and compared with a chi-squared test. No anti-HEV-3 IgG antibodies were detected in any of the samples from cats and dogs. Anti-HEV-3 IgG antibodies were detected in 2.82% of the serum samples from rabbits (2/71; 95% CI: 0.78-9.70). No significant correlations between seropositivity and gender, age, and symptoms (p > 0.05) were observed in rabbits. Our findings indicate that pet rabbits in Poland are exposed to HEV-3, develop humoral response due to infection and might constitute a source for HEV-3 transmission to humans.

Prevalence of hepatitis E virus genotype 4 of probable human origin in Tibetan pigs from the Qinghai-Tibetan Plateau, China.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In 2018-2022, we investigated the presence of HEV RNA in 1233 stool samples collected in the Qinghai-Tibetan Plateau, including humans (16), Tibetan pigs (624), yaks (312), sheep (267), and dogs (14). HEV RNA was only detected in Tibetan pig faecal samples (18.27%, 114/624). To perform molecular characterization of HEV strains in Tibetan pigs, we obtained 21 complete HEV genome sequences between 2018 and 2022. Sequence comparisons showed that 21 HEV strains from Tibetan pigs shared the mean nucleotide identities with the reference HEV strains ranging between 82.9% and 94.9% and 89.3% and 92.1% similarities with human HEV strains. Phylogenetic analysis confirmed that all HEV strains were genotype 4, closely related to human HEV strains. Sequence recombinant analysis showed five potential recombinant strains identified in this study, of which SWU/D18/2018 (GenBank No. MK410044) was recombinant with human and swine HEV strains, located 6509-6878 nt from the recombination point. Based on the Bayesian evolutionary trees, we found that most HEV strains diverged later than human HEV (16 Tibetan pig HEV strains diverged later than 1979, and seven human HEV strains diverged earlier than 1979). Therefore, we speculated that the prevalence of HEV 4 in Tibetan pigs possibly originated from humans in the Qinghai-Tibetan Plateau.

Anti-HEV seroprevalence and rate of viremia in a German cohort of dogs, cats, and horses.

Hepatitis E virus (HEV) genotype 3 infections in Germany are mainly transmitted zoonotically through the consumption of swine meat. Furthermore, there is evidence that pets might come into contact with HEV, but the relevance of companion animals as possible sources of HEV transmission in Germany still needs to be defined. A monitoring study was therefore carried out on dogs, cats, and horses from Germany. In total 365 serum samples from pets (124 dogs, 119 cats, and 122 horses) were tested for HEV by PCR and for anti-HEV antibodies by a commercial ELISA. The HEV seroprevalence determined by the sero-assay varied significantly between dogs (10%), cats (6%), and horses (2%). Liver injury-related enzymes, alanine transaminase (ALT), and aspartate transaminase (AST) showed no differences between HEV-positive or negative animals. None of the pet serum samples tested positive for PCR. This serological study suggests that dogs and cats are significantly exposed to HEV in Germany, while horses are of minor relevance.

Unraveling swine hepatitis E in the central region of Argentina through ELISA development and epidemiological insights.

Hepatitis E virus (HEV) is a public health concern globally, causing acute viral hepatitis in humans. Genotype-3 HEV (HEV-3), the most frequently genotype detected in South America, is zoonotic and the main reservoirs are the domestic pig and wild boar. Circulation of HEV-3 in Argentina has been confirmed in humans as well as in pig herds, wild boar and environmental waters. However, data are scarce mainly due to the inaccessibility of serological assays in this country. In order to provide insights in the epidemiology of HEV in swine in Argentina, we developed an indirect ELISA based on the native recombinant protein ORF2 and conducted a serological survey to determine the prevalence of seropositive swine in small-scale pig farms in the central region of Argentina. The method was evaluated in a panel of 157 serum samples, resulting in relative sensitivity of 98.6 % (95 % CI 95 %-100 %) and relative specificity of 97.7 % (95 % CI 94 %-100 %) compared to a commercial test. An almost perfect agreement was obtained between the two tests (Kappa index of 0.961). A survey on 294 samples from 49 small-scale farms resulted in a seropositivity rate of 54 %. Seropositive animals were found in 34 out of 49 (69.4 %) farms. Most of the farms (70.6 %) had over 50 % of seropositive animals. The wide spreading of HEV in the swine population of Tandil, Argentina, underscore the need to better understand the epidemiology of HEV in the region, enabling the implementation of targeted interventions to mitigate the impact of this virus on public health.

Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy.

Hepatitis E virus (HEV) is an important cause of acute viral hepatitis in humans worldwide. The food-borne transmission of HEV appears to be a major route in Europe through the consumption of pork and wild boar meat. HEV epidemiology in wild boars has been investigated mainly in Northern and Central Italian regions, whilst information from Southern Italy is limited. We investigated the occurrence of HEV in wild boar in the Apulia and Basilicata regions (Southern Italy). Thirteen (10.4%) out of one hundred and twenty-five wild boar samples tested positive for HEV using a quantitative reverse transcription PCR. HEV prevalence was 12% in Apulia and 9.3% in Basilicata. Seven samples were genotyped, and different subtypes (c, f, m) of genotype 3 were identified. The complete genome of a 3m strain was determined, and the virus showed the highest nucleotide identity to a human HEV strain identified in France in 2017. These findings demonstrate the substantial circulation of HEV in the wild boar population in Italian Southern regions. Gathering information on the HEV strains circulating in different geographical areas is useful for tracking the origin of HEV outbreaks and assessing the epidemiological role of wild boar as a potential virus reservoir for domestic pigs.

Surveillance for rat hepatitis E in wastewater networks, Italy.

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.

First Seroepidemiological Investigation of Hepatitis E Virus Infection in Backyard Pigs from Northeastern India: Prevalence and Associated Risk Factors.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis globally, with zoonotic potential, and pigs are considered the major reservoir. To determine the seroprevalence of HEV infection in pigs reared in backyard conditions in the northeastern region of India, blood samples were collected from 400 pigs from five northeastern states (80 samples from each state) and tested for IgG antibodies against HEV using an ELISA assay. Questionnaires on farm characteristics and management practices were completed, and risk factors associated with HEV were studied using univariate and multivariate analysis. The apparent seroprevalence of HEV infection was 51% (46.1-55.9, 95% CI), with a true prevalence of 52.98% (47.22-58.75, 95% CI). The risk factors significantly associated with higher HEV seropositivity were as follows: lack of disinfection (OR 4.65), feeding swill (restaurant and bakery waste) (OR 2.55), failure to follow the all-in-all-out production system (OR 3.47), and medium holding size (OR 9.83), which refers to mixed rearing of younger and older age groups. This study demonstrates that HEV is widespread among pigs reared in northeastern India. The risk factor analysis conducted in this study provides valuable insights into the prevalence of HEV in the region.

High circulation of hepatitis E virus (HEV) in pigs from the central region of Argentina without evidence of virus occurrence in pork meat and derived products.

Hepatitis E virus (HEV) is an emerging cause of viral hepatitis and pigs are considered a reservoir for the virus. HEV genotype 3 (HEV-3) has been reported in pigs, environmental matrices, and sporadic human cases in Argentina. We aimed to investigate HEV circulation in pigs from central Argentina and to assess the virus presence in pork meat and food products. Four types of samples obtained or derived from pigs collected in Córdoba province (Argentina) between 2019 and 2022, were tested: 276 serum samples were analyzed for anti-HEV antibody detection; stool (n = 20), pork meat (n = 71), and salami (n = 76) samples were studied for RNA-HEV detection, followed by sequencing and phylogenetic analyses. The positivity rate for anti-HEV antibodies was 80.1% (221/276). Eleven fecal samples (11/20) tested positive for RNA-HEV, from animals under 120 days of age. Three samples could be sequenced, and phylogenetic analyses revealed that they belonged to HEV-3 clade abchijklm, clustering close to strains previously detected in wastewater from Córdoba. None of the muscle meat or salami samples tested positive. A high HEV circulation in pigs was found, showing that these animals may play a significant role in the viral maintenance in the region, becoming a potential risk to the exposed population. Despite not detecting RNA-HEV in pork meat and salami in our study, we cannot rule out the possibility of foodborne transmission in Córdoba province.

Detection of Hepatitis E Virus in Rabbits and Rabbit Meat from Slaughterhouses in Hebei Province of China.

Background: Hepatitis E virus (HEV) is a zoonotic pathogen. HEV has been found to be widely prevalent in rabbits. Its isolates are classified into HEV-3, rabbit subgenotype (HEV-3ra). The routes of human infection with HEV-3ra remain unclear; however, foodborne transmission is possible when asymptomatically infected animals enter the food chain. The prevalence of HEV infection in slaughtered rabbits and the presence of HEV in rabbit meat were evaluated in this study. Materials and Methods: In three slaughterhouses in Hebei province, China, samples of rabbit blood were collected during the slaughter process, and muscle, liver, and cavity juice were collected from the rabbit carcasses. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay. HEV RNA was tested in all samples by reverse transcription nested PCR (RT-nested PCR). The final amplicons of RT-nested PCR were sequenced and phylogenetically analyzed. Results: Of the 459 serum samples, 50 [10.9%, 95% confidence interval (CI): 8.1-13.7] were positive for anti-HEV antibody, and 17 (3.7%, 95% CI: 2.0-5.4) were positive for HEV RNA. HEV RNA was detected in 7 of 60 liver samples (11.7%, 95% CI: 3.3-20) and 2 cavity juice samples from semi-eviscerated carcasses, but was not detected in any muscle sample from either the eviscerated or semi-eviscerated carcasses. All the detected HEV strains belonged to HEV-3ra and related most closely with the rabbit HEV sequence previously reported in China. Conclusion: A portion of rabbits were in the viremia period of HEV infection at the slaughter age, resulting in the possibility of HEV carriage by rabbit carcass, particularly semi-eviscerated carcass containing liver. These findings suggest a potential risk of HEV transmission from raw rabbit products entering the food chain, whereas the presence of HEV appeared to be lower in the eviscerated carcass than in the semi-eviscerated carcass.

Rat Hepatitis E Virus in Norway Rats, Ontario, Canada, 2018-2021.

We tested liver samples from 372 Norway rats (Rattus norvegicus) from southern Ontario, Canada, during 2018-2021 to investigate presence of hepatitis E virus infection. Overall, 21 (5.6%) rats tested positive for the virus. Sequence analysis demonstrated all infections to be rat hepatitis E virus (Rocahepevirus ratti genotype C1).

Hepatitis E Virus in the Iberian Peninsula: A Systematic Review.

One of the most frequent causes of acute viral hepatitis is hepatitis E virus (HEV) causing 20 million infections worldwide each year and 44,000 deaths. Studies on HEV in the Iberian Peninsula have been increasing through time with HEV infection being identified in humans and animals. The aim of the present systematic review was to compile and evaluate all the published data on HEV from studies performed in humans, animals and environmental samples in the Iberian Peninsula. The electronic databases Mendeley, PubMed, Scopus, and Web of Science were thoroughly searched, and research published up until February 01, 2023 were included. Resulting in a total of 151 eligible papers by full reading and application of PRISMA exclusion/inclusion criteria. Overall, the present review shows that several HEV genotypes, namely HEV-1, 3, 4, and 6 as well as Rocahepevirus, are circulating in humans, animals, and in the environment in the Iberian Peninsula. HEV-3 was the most common genotype circulating in humans in Portugal and Spain, as expected for developed countries, with HEV-1 only being detected in travelers and emigrants from HEV endemic regions. Spain is the biggest pork producer in Europe and given the high circulation of HEV in pigs, with HEV-3 being primarily associated to zoonotic transmission through consumption of swine meat and meat products, in our opinion, the introduction of an HEV surveillance system in swine and inclusion of HEV in diagnostic routines for acute and chronic human hepatitis would be important. Additionally, we propose that establishing a monitoring mechanism for HEV is crucial in order to gain a comprehensive understanding of the prevalence of this illness and the various strains present in the Iberian Peninsula, as well as their potential impact on public health.

Infection Dynamics and Genomic Mutations of Hepatitis E Virus in Naturally Infected Pigs on a Farrow-to-Finish Farm in Japan: A Survey from 2012 to 2021.

Hepatitis E virus (HEV) causes acute or chronic hepatitis in humans. Pigs are the primary reservoir for zoonotic HEV genotypes 3 and 4 worldwide. This study investigated the infection dynamics and genomic mutations of HEV in domestic pigs on a farrow-to-finish pig farm in Japan between 2012 and 2021. A high prevalence of anti-HEV IgG antibodies was noted among pigs on this farm in 2012, when the survey started, and persisted for at least nine years. During 2012-2021, HEV RNA was detected in both serum and fecal samples, indicating active viral replication. Environmental samples, including slurry samples in manure pits, feces on the floor, floor and wall swabs in pens, and dust samples, also tested positive for HEV RNA, suggesting potential sources of infection within the farm environment. Indeed, pigs raised in HEV-contaminated houses had a higher rate of HEV infection than those in an HEV-free house. All 104 HEV strains belonged to subgenotype 3b, showing a gradual decrease in nucleotide identities over time. The 2012 (swEJM1201802S) and 2021 (swEJM2100729F) HEV strains shared 97.9% sequence identity over the entire genome. Importantly, the swEJM2100729F strain efficiently propagated in human hepatoma cells, demonstrating its infectivity. These findings contribute to our understanding of the prevalence, transmission dynamics, and genetic characteristics of HEV in domestic pigs, emphasizing the potential risks associated with HEV infections and are crucial for developing effective strategies to mitigate the risk of HEV infection in both animals and humans.

First Insight into the Seroepidemiology of Hepatitis E Virus (HEV) in Dogs, Cats, Horses, Cattle, Sheep, and Goats from Bulgaria.

In recent years, hepatitis E virus (HEV) infection has been found to be widespread among different animal species worldwide. In Bulgaria, high HEV seropositivity was found among pigs (60.3%), wild boars (40.8%), and East Balkan swine (82.5%). The aim of the present study was to establish the seroprevalence of HEV among dogs, cats, horses, cattle, sheep, and goats in Bulgaria. In total, 720 serum samples from six animal species were randomly collected: dogs-90 samples; cats-90; horses-180; cattle-180; sheep-90; and goats-90. The serum samples were collected from seven districts of the country: Burgas, Kardzhali, Pazardzhik, Plovdiv, Sliven, Smolyan, and Stara Zagora. The animal serum samples were tested for HEV antibodies using the commercial Wantai HEV-Ab ELISA kit (Beijing, China). The overall HEV seroprevalence among different animal species from Bulgaria was as follows: dogs-21.1%; cats-17.7%; horses-8.3%; cattle-7.7%; sheep-32.2%; and goats-24.4%. We found the lowest overall HEV seropositivity in Plovdiv district (6.2%; 4/64; p = 0.203) and Smolyan district (8.8%; 4/45; p = 0.129), vs. the highest in Pazardzhik district (21.6%; 29/134; p = 0.024) and Burgas district (28.8%; 26/90; p = 0.062). To the best of our knowledge, this is the first serological evidence of HEV infection in dogs, cats, horses, cattle, sheep, and goats from Bulgaria. We found high HEV seropositivity in small ruminants (sheep and goats), moderate seropositivity in pets (dogs and cats), and a low level of seropositivity in large animals (horses and cattle). Previous Bulgarian studies and the results of this research show that HEV infection is widespread among animals in our country. In this regard, the Bulgarian health authorities must carry out increased surveillance and control of HEV infection among animals in Bulgaria.

Low Replication Efficiency of a Japanese Rabbit Hepatitis E Virus Strain in the Human Hepatocarcinoma Cell Line PLC/PRF/5.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

First Detection of Hepatitis E Virus (Rocahepevirus ratti Genotype C1) in Synanthropic Norway Rats (Rattus norvegicus) in Romania.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with different viral genera and species reported in a wide range of animals. Rodents, particularly rats, carry the specific genus rat HEV (Rocahepevirus genus, genotype C1) and are exposed occasionally to HEV-3 (Paslahepevirus genus, genotype 3), a zoonotic genotype identified in humans and widely distributed in domestic and feral pigs. In this study, the presence of HEV was investigated in synanthropic Norway rats from Eastern Romania, in areas where the presence of HEV-3 was previously reported in pigs, wild boars and humans. Using methods capable of detecting different HEV species, the presence of HEV RNA was investigated in 69 liver samples collected from 52 rats and other animal species. Nine rat liver samples were identified as being positive for rat HEV RNA (17.3%). High sequence identity (85-89% nt) was found with other European Rocahepevirus. All samples tested from other animal species, within the same environment, were negative for HEV. This is the first study to demonstrate the presence of HEV in rats from Romania. Since rat HEV has been reported to cause zoonotic infections in humans, this finding supports the need to extend the diagnosis of Rocahepevirus in humans with suspicion of hepatitis.