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.

Genomic and spatial variability of a European common vole hepevirus.

Rodents host different orthohepeviruses, namely orthohepevirus C genotype HEV-C1 (rat hepatitis E virus, HEV) and the additional putative genotypes HEV-C3 and HEV-C4. Here, we screened 2,961 rodents from Central Europe by reverse transcription polymerase chain reaction (RT-PCR) and identified HEV RNA in 13 common voles (Microtus arvalis) and one bank vole (Myodes glareolus) with detection rates of 2% (95% confidence interval [CI]: 1-3.4) and 0.08% (95% CI: 0.002-0.46), respectively. Sequencing of a 279-nucleotide RT-PCR amplicon corresponding to a region within open reading frame (ORF) 1 showed a high degree of similarity to recently described common vole-associated HEV (cvHEV) sequences from Hungary. Five novel complete cvHEV genome sequences from Central Europe showed the typical HEV genome organization with ORF1, ORF2 and ORF3 and RNA secondary structure. Uncommon features included a noncanonical start codon in ORF3, multiple insertions and deletions within ORF1 and ORF2/ORF3, and the absence of a putative ORF4. Phylogenetic analysis showed all of the novel cvHEV sequences to be monophyletic, clustering most closely with an unassigned bird-derived sequence and other sequences of the species Orthohepevirus C. The nucleotide and amino acid sequence divergence of the common vole-derived sequences was significantly correlated with the spatial distance between the trapping sites, indicating mostly local evolutionary processes. Detection of closely related HEV sequences in common voles in multiple localities over a distance of 800 kilometers suggested that common voles are infected by cvHEV across broad geographic distances. The common vole-associated HEV strain is clearly divergent from HEV sequences recently found in narrow-headed voles (Microtus gregalis) and other cricetid rodents.

Avian hepatitis E virus is widespread among chickens in Poland and belongs to genotype 2.

Big liver and spleen disease, caused by avian hepatitis E virus, has been reported in Poland, but the prevalence of the virus has not yet been investigated. In this study, 1034 serum samples from 57 breeder broiler and laying hen flocks were screened for the presence of anti-aHEV antibodies. In a random serology study, 56.1% of flocks were positive. Seroprevalence was higher in laying hen flocks than in broiler breeder flocks. Phylogenetic analysis of partial ORF1 and ORF2 sequences revealed that all Polish isolates belonged to genotype 2. This is the first time this genotype has been detected in Central Europe.

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs.

BACKGROUND: Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings. OBJECTIVE: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of NGMS compared with quantitative clinical standards. DESIGN: Longitudinal and cross-sectional studies. SETTING: A clinical trial (ClinicalTrials.gov: NCT01285050) and a well-characterized cohort study of persons who have injected drugs. PARTICIPANTS: Persons co-infected with hepatitis C virus (HCV) and HIV. MEASUREMENTS: Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR). RESULTS: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained more than 10 000 copies/mL or IU/mL, respectively, as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1), was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Through use of a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs. In these persons, HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as HCV RNA and SEN virus D. LIMITATION: The medical importance of HHpgV-1 infection is unknown. CONCLUSION: Although NGMS is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as HHpgV-1. PRIMARY FUNDING SOURCE: National Institutes of Health.

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.

Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.

BACKGROUND: Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens. RESULTS: Based on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/µl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens. CONCLUSIONS: A rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Avian Hepatitis E Virus Infection in Organic Layers.

Between 2012 and 2014, 141 chickens from 10 organic layer flocks with a history of severe drop in egg production (up to 40%) and slight increased mortality (up to 1% per week) were submitted to the Avian Health and Food Safety Laboratory (Puyallup, WA). At necropsy, the most common finding was pinpoint white foci on the liver and regressed ova without any other remarkable lesions. Histologically, there was multifocal mild-to-severe acute necrotizing hepatitis present. No significant bacteria were recovered from liver samples, and tests for mycotoxins were negative. Twenty-six serum samples from four affected flocks tested were positive for avian hepatitis E virus (HEV) immunoglobulin Y antibodies. Avian HEV RNA was detected in 10 livers of chickens from two different affected flocks. The avian HEV was characterized by sequencing and determined to belong to genotype 2. The diagnosis of a clinical manifest HEV was based solely on the demonstration of specific viral RNA and the absence of other causative agents in samples from flocks, as the clinical sings and pathologic lesions were atypical.

Hepatitis E infection in HIV-infected liver and kidney transplant candidates.

Hepatitis E virus (HEV) has been reported to cause acute and chronic hepatitis in those with HIV infection and among solid organ transplant recipients in Europe. Limited data indicate that HEV is endemic in the United States, but the prevalence and significance of HEV infection among those with HIV and awaiting solid organ transplantation is unknown. We evaluated anti-HEV IgM and IgG antibodies and HEV RNA in 166 HIV-infected solid organ transplant candidates enrolled in the NIH HIV-Transplant Cohort. Overall prevalence of anti-HEV IgG approached 20% in both liver and renal transplant candidates. Evidence of recent infection was present in approximately 2% of liver transplant candidates and none of the kidney transplant candidates. HEV RNA was not detected in any patient. We conclude that markers of HEV infection are frequent among candidates for transplantation, but active, ongoing viremia is not seen. Evidence of recent infection (acute on chronic) liver disease was present in liver but not kidney recipients.

Hepatitis E in liver transplant recipients in the Rhône-Alpes region in France.

PURPOSE: In developed countries, hepatitis E virus (HEV) is considered an emerging pathogen, but prevalence seems highly variable according to previous European studies. As HEV can lead to chronic infections in immunosuppressed patients, it is thus essential to evaluate the prevalence and incidence of this infection. METHODS: We determined retrospectively, in a cohort of 206 pediatric and adult liver transplant recipients from the Rhône-Alpes region in France, pre-transplant anti-HEV-IgG prevalence and incidence of HEV infections during post-transplant follow-up (HEV IgG and IgM ± HEV-RNA). RESULTS: Transplantations were carried out between 2005 and 2012 and mean post-transplant follow-up was 32.8 months. Global pre-transplant prevalence of anti-HEV IgG was 29%, increasing regularly with age from 7% for children under 15 to 49% for patients older than 60. From the 142 seronegative patients before transplant, 11 seroconversions (7.7%) were observed during follow-up (incidence of 2.83 cases per 100 person-years). HEV RNA-tested at transaminases peak or randomly-was detected in only one case of seroconversion. For at least 2 HEV-seropositive patients, who had negative RNAemia before transplantation, viral RNA was detected chronically during follow-up, suggesting reinfection with HEV. CONCLUSION: Acute infections were largely more frequent than chronic infections and were asymptomatic or misdiagnosed, suggesting that liver transplant patients may not be particularly prone to developing severe HEV hepatitis. In addition, the presence of IgG anti-HEV may not protect against re-infection. Serological testing, therefore, appears to be of limited interest for the diagnosis of HEV infections in liver transplant recipients.

Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus.

In a prospective longitudinal study, a broiler breeder flock and its progeny were monitored for the presence of avian hepatitis E virus (HEV) RNA and antibodies. The flock was part of a multiple-age farm where the presence of avian HEV with clinical signs (increased mortality and decreased egg production) was demonstrated in several previous production cycles. Samples were taken twice at the rearing site and several times at the production site from broiler breeders including cockerels and day-old chicks. The samples were investigated by conventional and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and histological methods. At all time points, samples from the hens were positive for avian HEV RNA. The birds did not show any clinical signs, even though histopathological lesions of non-specific aetiology in the liver and spleen could be demonstrated. A significant increase in the number of positive birds and viral load was seen in week 45, in accordance with an increase in antibody titres. In comparison, cockerels investigated in week 62 tested negative by RT-PCR and ELISA. Avian HEV RNA was also detected in day-old chicks hatched from eggs laid in week 25, indicating vertical transmission. All partial helicase and capsid sequences retrieved within this study clustered together and were identical to previous sequences obtained from the same multiple-age farm. In conclusion, avian HEV persisted on the farm over years and circulated between the rearing and the production sites without causing any clinical signs although high viral loads in the adult hens were observed.

Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations.

Two commercial Midwestern egg-type chicken flocks experienced significant increases in mortality rates in April 2013 with clinical signs appearing in 17-week-old pullets on Farm A and in 46-week-old hens on Farm B. Average weekly mortality was 0.44% over a 4-week period on Farm A and 0.17% over an 8-week period on Farm B. On Farm A, flocks in the affected house had a 45% decrease in daily egg production from weeks 19 to 27 when compared with standard egg production curves (P < 0.01) while no decrease in egg production was noticed on Farm B. Post-mortem examination revealed changes consistent with hepatitis-splenomegaly syndrome, including hepatomegaly with serosanguineous fluid in the coelomic cavity and hepatic subcapsular haemorrhages. Microscopic lesions were characterized by multifocal necrotizing hepatitis and intrahepatic haemorrhage. No significant bacteria were recovered from liver samples, but 72 to 100% of the liver samples from affected chickens on Farm A (8/11) and Farm B (7/7) contained detectable amounts of avian hepatitis E virus (aHEV) RNA as determined by polymerase chain reaction. Sequencing and phylogenetic analysis of a 361-base-pair fragment of the helicase gene demonstrated 98.6 to 100% nucleotide identity between the aHEV genomes from Farm A and Farm B, whereas identities ranged from 74.6 to 90.5% when compared with other representative sequences. Sequences from this study clustered within aHEV genotype 2 previously recognized in the USA. In contrast to other reported aHEV outbreaks that occurred in 30-week-old to 80-week-old chickens, in the present investigation clinical aHEV was identified in 17-week-old chickens on one of the farms.

Pathogenicity of two different genotypes avian hepatitis E strains in laying hens and silkie fowl.

To determine the pathogenicity of two different genotypes of avian hepatitis E strains in two species of birds, a total of thirty healthy 12-week-old birds were used. After inoculation, fecal virus shedding, viremia, seroconversion, serum alanine aminotransferase (ALT) increases and liver lesions were evaluated. The results revealed that CHN-GS-aHEV and CaHEV could both infect Hy-Line hens and silkie fowls, respectively. Compared to the original avian HEV strain, the cross-infected virus exhibited a delay of 2 weeks and 1 week in emerged seroconversion, viremia, fecal virus shedding, and increased ALT level, and also showed mild liver lesions. These findings suggested that CHN-GS-aHEV may have circulated in chickens. Overall, these two different genotypes of avian HEV showed some variant pathogenicity in different bird species. This study provides valuable data for further analysis of the epidemic conditions of two avian HEVs in Hy-Line hens and silkie fowls.

Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis in tropical and temperate climates. Tropical genotypes 1 and 2 are associated with food-borne and waterborne transmission. Zoonotic reservoirs (mainly pigs, wild boar, and deer) are considered for genotypes 3 and 4, which exist in temperate climates. In view of the association of several zoonotic viruses with bats, we analyzed 3,869 bat specimens from 85 different species and from five continents for hepevirus RNA. HEVs were detected in African, Central American, and European bats, forming a novel phylogenetic clade in the family Hepeviridae. Bat hepeviruses were highly diversified and comparable to human HEV in sequence variation. No evidence for the transmission of bat hepeviruses to humans was found in over 90,000 human blood donations and individual patient sera. Full-genome analysis of one representative virus confirmed formal classification within the family Hepeviridae. Sequence- and distance-based taxonomic evaluations suggested that bat hepeviruses constitute a distinct genus within the family Hepeviridae and that at least three other genera comprising human, rodent, and avian hepeviruses can be designated. This may imply that hepeviruses invaded mammalian hosts nonrecently and underwent speciation according to their host restrictions. Human HEV-related viruses in farmed and peridomestic animals might represent secondary acquisitions of human viruses, rather than animal precursors causally involved in the evolution of human HEV.

Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea.

Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57 % of chicken flocks and 28 % of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97 % nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.

Avian hepatitis E virus identified in Russian chicken flocks exhibits high genetic divergence based on the ORF2 capsid gene.

A total of 79 liver samples from clinically sick and asymptomatic chickens were tested for avian hepatitis E virus (aHEV). Samples were received from 19 farms, five of which tested positive with primers targeting the ORF2 capsid gene. The phylogenetic analysis of a 242-base-pair fragment demonstrated that the Russian aHEV isolates share between 78.2 and 96.2% over the fragment sequenced, whereas the nucleotide sequence identities between the Russian isolates and the other representatives from GeneBank varied from 76.3 to 96.2%. The homology between the studied hepatitis E viruses and swine hepatitis E virus varied between 46.9 to 48.1%. The most divergent isolate aHEV16050 showed homology of 82.6% as compared with the strains in the dendrogram. The three positive hepatitis E virus samples (aHEV16279, aHEV16050 and aHEV18196) did not cluster with the European genotype 3 as expected due to the close location of Russia to Europe, nor did they with the other two genotypes, separating to a distinct branch. The aHEV16211 grouped together with European and Chinese isolates, and the aHEV18198 with Canadian ones.

[Detection and description of avian hepatitis E virus isolated in China--a review].

Avian hepatitis E virus (HEV), a member of Hepeviridae family, is genetically and antigenically related with human and swine HEV in the family. Since its discovery, avian HEV infection has been investigated in many countries from serology and molecular epidemiology studies. At present, five complete or near complete genomes of avian HEV isolates were reported in GenBank and were divided into three genotypes. The complete genome of avian HEV contains 3 ORFs of which ORF2 gene encodes capsid protein containing the primary epitopes of viral particles and is target gene for serodiagnostic antigen and vaccine candidate. Because avian HEV infection has significant impact on the poultry industry and potential zoonotic transmission, the researches on avian HEV have been given much attention. We here give a broad review of the research update on the aetiology, pathogenesis and the antigenicity of capsid protein of avian HEV based on identification of Chinese avian HEV isolate.

TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

Identification of a Putative Novel Genotype of Avian Hepatitis E Virus from Apparently Healthy Chickens in Southwestern Nigeria.

Avian hepatitis E virus (aHEV) is the major etiological agent of hepatitis-splenomegaly syndrome (HSS), big liver and spleen disease (BLSD), and hepatic rupture hemorrhage syndrome (HRHS) in chickens. Infections with aHEV cause a significant decrease in egg production and increased mortality in chickens worldwide. However, studies on the prevalence of aHEV in Nigeria are scarce. In this study, serum (n = 88) and fecal samples (n = 110) obtained from apparently healthy layer chickens from three states in southwestern Nigeria were analyzed by nested reverse transcription-polymerase chain reaction (nRT-PCR) targeting the helicase and capsid gene for the presence of aHEV. Avian HEV was detected in 12.5% (n = 11/88) of serum samples and 9.1% (n = 10/110) of fecal samples tested. Phylogenetic analysis showed that five of the twelve identified aHEV sequences belonged to genotype 2. The remaining seven sequences were only distantly related to other known aHEV isolates. After amplification of the near-complete ORF2 fragment (1618 bp) and part of the ORF1 (582 bp) of isolate YF40_aHEV_NG phylogenetic analysis revealed a nucleotide sequence identity between 79.0 and 82.6% and 80.1 and 83.5%, respectively, to other known aHEV strains, indicating that the Nigerian isolate YF40_aHEV_NG belongs to a novel aHEV genotype. This is the first report of co-circulation of aHEV genotypes in chickens in Nigeria.

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.