Isolation and characterization of a novel rodent hepevirus in long-tailed dwarf hamsters (Cricetulus longicaudatus) in China.

Hepeviruses have been identified in a broad range of animal hosts, including mammals, birds, and fish. In this study, rodents (n=91) from seven different species and ten pikas (Ochotona curzoniae) were collected in Qinghai Province, China. Using transcriptomic sequencing and confirmatory molecular testing, hepeviruses were detected in 27 of 45 (60 %) long-tailed dwarf hamsters (Cricetulus longicaudatus) and were undetected in other rodents and pika. The complete genome sequences from 14 representative strains were subsequently obtained, and phylogenetic analyses suggested that they represent a novel species within the genus Rocahepevirus, which we tentatively designated as Cl-2018QH. The virus was successfully isolated in human hepatoma (Huh-7) and murine fibroblast (17 Cl-1) cell lines, though both exhibited limited replication as assayed by detection of negative-sense RNA intermediates. A129 immunodeficient mice were inoculated with Cl-2018QH and the virus was consistently detected in multiple organs, despite relatively low viral loads. In summary, this study has described a novel rodent hepevirus, which enhances our knowledge of the genetic diversity of rodent hepeviruses and highlights its potential for cross-species transmission.

Intrinsically disordered regions in the rodent hepevirus proteome.

Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus's pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.

Genome characterization, prevalence and tissue distribution of astrovirus, hepevirus and norovirus among wild and laboratory rats (Rattus norvegicus) and mice (Mus musculus) in Hungary.

Rodents including rats are reservoir of several pathogens capable of affecting human health. In this study, faecal and different organ specimens from free-living Norway rats (Rattus norvegicus) (N = 18) and faecal samples from laboratory rodents (rats N = 21 and mice N = 20) collected from different geographic areas in Hungary between 2017 and 2020 were investigated by viral metagenomics and conventional RT-PCR methods. The complete genome of three different RNA viruses, rat astrovirus, rat norovirus and rat hepevirus were characterized and analysed in detail. Rat norovirus was detected in faecal (17.6%, 3/17) and kidney (7.1%, 1/14) samples; rat astrovirus in faecal (23.5%, 4/17) and spleen (13.3%, 2/15) samples, and rat hepevirus in 43% to 67% the faecal, liver, kidney, lung, heart, muscle, brain and blood samples from Norway rats, respectively. Rat norovirus was also identifiable in 5% (1/21) of laboratory rats and rat astrovirus in 40% (8/20) of faecal samples from laboratory mice. Co-infections were found in 28% (5/18) wild Norway rats. The highest RNA viral load of astrovirus (1.81 × 108 copy/g) and norovirus (3.49 × 107 copy/g) were measured in faecal samples; while the highest RNA viral load of hepevirus (1.16 × 109 copy/g) was found in liver samples of Norway rats, respectively. This study confirms the wide geographic distribution and high prevalence of astrovirus, norovirus and hepevirus among wild rats in Hungary with confirmation of different organ involvement of as well as the detection of norovirus and astrovirus in laboratory rats and mice, respectively. This finding further strengthens the role of rodents in the spread of viral pathogens especially infecting human.

Review of Hepatitis E Virus in Rats: Evident Risk of Species Orthohepevirus C to Human Zoonotic Infection and Disease.

Hepatitis E virus (HEV) (family Hepeviridae) is one of the most common human pathogens, causing acute hepatitis and an increasingly recognized etiological agent in chronic hepatitis and extrahepatic manifestations. Recent studies reported that not only are the classical members of the species Orthohepevirus A (HEV-A) pathogenic to humans but a genetically highly divergent rat origin hepevirus (HEV-C1) in species Orthohepevirus C (HEV-C) is also able to cause zoonotic infection and symptomatic disease (hepatitis) in humans. This review summarizes the current knowledge of hepeviruses in rodents with special focus of rat origin HEV-C1. Cross-species transmission and genetic diversity of HEV-C1 and confirmation of HEV-C1 infections and symptomatic disease in humans re-opened the long-lasting and full of surprises story of HEV in human. This novel knowledge has a consequence to the epidemiology, clinical aspects, laboratory diagnosis, and prevention of HEV infection in humans.

Aislamiento de una cepa genotipo 3 del virus de la hepatitis E obtenida de heces humanas / Cell culture isolation of hepatitis e virus genotype 3 strain obtained from human feces

ABSTRACT Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/µL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/µL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

RESUMEN El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/µL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/µL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV.

Genetically highly divergent RNA virus with astrovirus-like (5'-end) and hepevirus-like (3'-end) genome organization in carnivorous birds, European roller (Coracias garrulus).

Astroviruses (family Astroviridae) and hepeviruses (family Hepeviridae) are small, non-enveloped viruses with genetically diverse +ssRNA genome thought to be enteric pathogens infecting vertebrates including humans. Recently, many novel astro- and hepatitis E virus-like +ssRNA viruses have been described from lower vertebrate species. The non-structural proteins of astro- and hepeviruses are highly diverse, but the structural/capsid proteins represent a common phylogenetic position shed the light of their common origin by inter-viral recombination. In this study, a novel astrovirus/hepevirus-like virus with +ssRNA genome (Er/SZAL5/HUN/2011, MK450332) was serendipitously identified and characterized from 3 (8.5%) out of 35 European roller (Coracias garrulus) faecal samples by RT-PCR in Hungary. The complete genome of Er/SZAL5/HUN/2011 (MK450332) is 8402 nt-long and potentially composed three non-overlapping open reading frames (ORFs): ORF1a (4449 nt/1482aa), ORF1b (1206 nt/401aa) and ORF2 (1491 nt/496aa). The ORF1ab has an astrovirus-like genome organization containing the non-structural conserved elements (TM, CC, NLS, VPg) and enzyme residues (trypsine-like protease, RNA-dependent RNA-polymerase) with low amino acid sequence identity, 15% (ORF1a) and 44% (ORF1b), to astroviruses. Supposedly the ORF2 is a capsid protein but neither the astrovirus-like subgenomic RNA promoter (sgRNA) nor the astrovirus-like capsid characteristics have been identifiable. However, the predicted capsid protein (ORF2) showed 26% identity to the corresponding protein of hepevirus-like novel Rana hepevirus (MH330682). This novel +ssRNA virus strain Er/SZAL5/HUN/2011 with astrovirus-like genome organization in the non-structural genome regions (ORF1a and ORF1b) and Rana hepevirus-related capsid (ORF2) protein represent a potentially recombinant virus species and supports the common origin hypothesis, although, the taxonomic position of the studied virus is still under discussion.

Characterization of a quasi-enveloped, fast replicating hepevirus from fish and its use as hepatitis E virus surrogate.

Hepatitis E virus (HEV) is an emerging concern for the safety of plasma-derived medicinal products. The lack of an efficient cell culture system hampers the studies on HEV biology as well as validation studies to test the capacity of virus reduction steps to clear HEV. Hence, a surrogate hepevirus that can efficiently replicate in cell culture is needed. Cutthroat trout virus (CTV) is a non-pathogenic fish hepevirus, which can replicate in cell culture to high titers. Under interferon inhibition, CTV replication reached up to 5 × 107 genome equivalents per µL in 4-5 days. The intracellular CTV progeny was already lipid-associated, suggesting that the envelope is acquired from intracellular membranes. Transmission electron microscopy of purified quasi-enveloped virus revealed exosome-like structures with an average size of 40 nm, in contrast to 27-34 nm for the non-enveloped virus. The quasi-enveloped virus was significantly less infectious than the non-enveloped virus. Assays based on quantitative RT-PCR, immunofluorescence and immunocytochemistry were established to evaluate virus inactivation. Cold ethanol fractionation removed 3.0 log of CTV and pasteurization of human albumin inactivated more than 3.7 log to below the limit of detection. Similar to HEV, virus replication was promoted in the presence of 17ß-estradiol, an effect that can contribute to the understanding of the exacerbated virulence of HEV in pregnant women. These results together reveal substantial similarities between the human and fish HEV and validate CTV as a practical virus model to use in some applications for evaluating the HEV reduction capacity of biological manufacturing process steps.

Detection of bat hepatitis E virus RNA in microbats in Japan.

Several recent studies have reported that various bat species harbor bat hepatitis E viruses (BatHEV) belonging to the family Hepeviridae, which also contains human hepatitis E virus (HEV). The distribution and ecology of BatHEV are not well known. Here, we collected and screened 81 bat fecal samples from nine bat species in Japan to detect BatHEV RNA by RT-PCR using HEV-specific primers, and detected three positive samples. Sequence and phylogenetic analyses indicated that these three viruses were BatHEVs belonging to genus Orthohepevirus D like other BatHEV strains reported earlier in various countries. These data support the first detection of BatHEVs in Japanese microbats, indicating their wide geographical distribution among multiple bat species.

Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China.

BACKGROUND: In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China. METHODS: A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses. RESULTS: Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels. CONCLUSIONS: This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae.

Serological evidence of Hepatitis E Virus infection in Antioquia, Colombia slaughtered pigs / Evidencia serológica de infección por el Virus de Hepatitis E en cerdos faenados en Antioquia, Colombia

Objective. To detect the presence of specific antibodies against Hepatitis E virus (HEV) in pigs slaughtered in Antioquia, the department where the greatest amount of pork is produced and consumed in Colombia. Materials and methods. Between September 2011 and May 2012, blood samples from pigs were obtained in five slaughterhouses of Antioquia, four of them located in the Aburrá Valley subregion and other located in northern subregión. Serum were evaluated with a commercial ELISA kit for diagnosing HEV in humans but adapted to detect IgG and IgM antibodies in pigs. Results. A 100.0% seropositivity for IgG antibodies was found in 1000 samples evaluated, and 82.06% for IgM antibodies were found in 740 samples. Conclusions. These results indicate that pigs in slaughter age in Antioquia, and possibly in Colombia, have been exposed to HEV at some point in their production process and a high percentage of them can arrive to slaughterhouses with recent infection.

Objetivo. Detectar la presencia de anticuerpos específicos contra el virus de la Hepatitis E (HEV) en cerdos faenados en Antioquia, departamento donde se produce y consume la mayor cantidad de carne de cerdo en Colombia. Materiales y métodos. Entre septiembre de 2011 y mayo de 2012, se obtuvieron muestras de sangre de cerdos en cinco plantas de faenado, cuatro de ellas ubicadas en el Valle de Aburrá y una en la subregión Norte del departamento de Antioquia, las cuales fueron evaluadas mediante un kit de ELISA comercial para diagnóstico de HEV en humanos pero adaptado para la detección de anticuerpos tipo IgG e IgM en cerdos. Resultados. Se encontró una seropositividad de 100.0% para anticuerpos tipo IgG en 1000 muestras evaluadas y de 82.06% para anticuerpos tipo IgM en 740 muestras. Una muestra de heces positiva para la detección del genoma HEV es similar al genotipo 3 encontrada en Estados Unidos. Conclusiones. Estos resultados indican que los cerdos en edad de faenado en Antioquia y posiblemente en Colombia, han tenido exposición a HEV del, un virus zoonótico emergente a nivel mundial, en algún momento de su proceso productivo.