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.

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.

Hepatitis E virus causes apoptosis of ovarian cells in hens and resulting in a decrease in egg production.

Previous studies have shown that avian hepatitis E virus (HEV) decreases egg production by 10-40% in laying hens, but have not fully elucidated the mechanism of there. In this study, we evaluated the replication of avian HEV in the ovaries of laying hens and the mechanism underlying the decrease in egg production. Forty 150-days-old commercial laying hens were randomly divided into 2 groups of 20 hens each. A total of 1 mL (104GE) of avian HEV stock was inoculated intravenously into each chicken in the experimental group, with 20 chickens in the other group serving as negative controls. Five chickens from each group were necropsied weekly for histopathological examination. The pathogenicity of avian HEV has been characterized by seroconversion, viremia, fecal virus shedding, ovarian lesions, and decreased egg production. Both positive and negative-strand avian HEV RNA, and ORF2 antigens can be detected in the ovaries, suggesting that avian HEV can replicate in the ovaries and serve as an important extrahepatic replication site. The ovaries of laying hens underwent apoptosis after avian HEV infection. These results indicate that avian HEV infection and replication in ovarian tissues cause structural damage to the cells, leading to decreased egg production.

FAdV-4 Promotes Expression of Multiple Cytokines and Inhibits the Proliferation of aHEV in LMH Cells.

Single or mixed infections of multiple pathogens such as avian hepatitis E virus (aHEV) and avian leukosis virus subgroup J (ALV-J) have been detected in numerous laying hens with severe liver injury in China. Thus, aHEV and immunosuppressive viruses are speculated to cause co-infections. In this study, co-infection with aHEV and fowl adenovirus (FAdV) was confirmed by nested RT-PCR and recombinase-aided amplification combined with gene sequencing in two flocks with severe liver injury. Subsequently, the two reference strains, aHEV and FAdV-4, were inoculated into LMH cells to identify their co-infection potential. Confocal microscopy revealed aHEV and FAdV-4 co-infected LMH cells. In addition, the replication dynamics of aHEV and FAdV-4 along with the expression levels of immuno-cytokines were measured. The results indicated colocalization of aHEV and FAdV-4 and inhibition of viral replication in LMH cells. The transcription levels of MDA5, Mx, OASL, and IFN-α were significantly upregulated in LMH cells, whereas those of immune-related factors induced by FAdV-4 were downregulated upon FAdV-4 and aHEV co-infection. These results confirmed the co-infection of aHEV and FAdV-4 in vitro and prompted the antagonistic pathogenic effects of FAdV-4 and aHEV, thereby providing novel insights into the counterbalancing effects of these viruses.

Inflammatory response triggered by avian hepatitis E virus in vivo and in vitro.

Hepatitis E virus (HEV) is relevant to public health worldwide, and it affects a variety of animals. Big liver and spleen disease (BLS) and hepatitis-splenomegaly syndrome (HSS) associated with avian HEV (aHEV) were first reported in 1988 and in 1991, respectively. Here, cell culture-adapted aHEV genotype 3 strain, YT-aHEV (YT strain), a typical genotype isolated in China, was used for basic and applied research. We evaluated liver injury during the early stages of infection caused by the YT strain in vivo. Both in vivo and in vitro experimental data demonstrated that viral infection induces innate immunity, with mRNA expression levels of two key inflammatory factors, interleukin-1ß (IL-1ß) and IL-18, significantly upregulated. The YT strain infection was associated with the activation of Toll-like receptors (TLRs), nuclear factor kappa B (NF-κB), caspase-1, and NOD-like receptors (NLRs) in the liver and primary hepatocellular carcinoma epithelial cells (LMH). Moreover, inhibiting c-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK1 or 2), P38, NF-κB, or caspase-1 activity has different effects on NLRs, and there is a mutual regulatory relationship between these signaling pathways. The results show that SB 203580, U0126, and VX-765 inhibited IL-1ß and IL-18 induced by the YT strain, whereas Pyrrolidinedithiocarbamate (PDTC) had no significant effect on the activity of IL-1ß and IL-18. Pretreatment of cells with SP600125 had an inhibitory effect on IL-18 but not on IL-1ß. The analysis of inhibition results suggests that there is a connection between Mitogen-activated protein kinase (MAPK), NF-κB, and the NLRs signaling pathways. This study explains the relationship between signaling pathway activation (TLRs, NF-κB, MAPK, and NLR-caspase-1) and viral-associated inflammation caused by YT strain infection, which will help to dynamic interaction between aHEV and host innate immunity.

Combined inhibitory effect of interferon and antiserum on avian hepatitis E virus.

Epidemiologic investigations in recent years have shown that the detection rate of avian hepatitis E virus (HEV) in chicken flocks is increasing in China. Nevertheless, effective prevention and control measures are still lacking. In this study, specific pathogen-free (SPF) chicken serum against HEV was prepared using recombinant HEV open reading frames (ORF2 and ORF3) proteins as immunogens. An SPF chicken infection model was established by intravenous inoculation of chick embryos. Swab samples were collected at 7, 14, 21, and 28 d of age and used to detect avian HEV load, along with other indicators, by fluorescence quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. The therapeutic effects on blocking vertical HEV transmission were observed, by using the methods of antibody application alone, mixed, or combined application of each of the 2 antibodies with type I interferon. The results showed that type I interferon alone or in combination with antiserum reduced the positive rate of HEV from 100 to 62.5% and 25%, respectively. However, the avian HEV-positivity rate was reduced to 75, 50, and 37.5% after type I interferon was used alone or in combination with antisera against ORF2 and ORF3, respectively. The inhibitory effect of type I interferon alone or in combination with an antiserum, on HEV replication was more significant in cells than in vivo. In this study, the inhibitory effect of type I interferon alone or in combination with an antiserum on avian HEV replication was observed in vitro and in vivo, providing the necessary technical reserve for disease prevention and control.

Development of a novel competitive ELISA based on nanobody-horseradish peroxidase fusion protein for rapid detection of antibodies against avian hepatitis E virus.

Avian hepatitis E virus (avian HEV) increases poultry mortality and decreases egg production, leading to huge economic losses worldwide. However, there is no effective serological test for avian HEV. Researchers previously created a testing platform using the nanobody (Nb)-horseradish peroxidase (HRP) fusion protein as an ultrasensitive probe to develop competitive ELISA (cELISA) to detect antibodies against different animal viruses. In this study, a rapid and reliable cELISA was developed to test for antibodies against avian HEV using the same platform. Six anti-avian HEV capsid protein nanobodies were selected from an immunized Bactrian camel using phage display technology. The avian HEV-Nb49-HRP fusion protein was expressed and used as a probe for developing a cELISA assay to test for avian HEV antibodies. The cut-off value of the developed cELISA was 22.0%. There was no cross-reaction with other anti-avian virus antibodies, suggesting that the cELISA had good specificity. The coefficients of variation were 0.91% to 4.21% (intra-assay) and 1.52% to 6.35% (inter-assay). Both cELISA and indirect ELISA showed a consistency of 86.7% (kappa = 0.738) for clinical chicken serum samples, and coincidence between cELISA and Western blot was 96.0% (kappa = 0.919). The epitope recognized by Nb49 was located in aa 593-604 of the avian HEV capsid protein, and the peptide (TFPS) in aa 601-604 was essential for binding. The novel cELISA is a saving cost, rapid, useful, and reliable assay for the serological investigation of avian HEV. More importantly, the peptide TFPS may be crucial to immunodominant antigen composition and protection.

A novel genotype of avian hepatitis E virus identified in chickens and common pheasants (Phasianus colchicus), extending its host range.

In 2019, outbreaks of hepatitis-splenomegaly syndrome (HSS) were observed in six commercial layer chicken flocks, belonging to three different Polish farms, and characterized by increased mortality, hemorrhagic hepatitis with attached blood clots on the liver surface, and splenomegaly. Diseased flocks were initially investigated for the presence of avian hepatitis E virus (aHEV) - the etiological agent of HSS - by conventional reverse transcriptase polymerase chain reaction, which revealed aHEV sequences clustering separately from all known aHEV genotypes. Additionally, an aHEV genome was identified for the first time in common pheasants, from a flock in France, using Next Generation Sequencing. This genome clustered together with the Polish aHEVs here investigated. Complete genome aHEV sequences from the HSS outbreaks confirmed the divergent cluster, with a shared nucleotide sequence identity of 79.6-83.2% with other aHEVs, which we propose to comprise a novel aHEV genotype - genotype 7. Histology and immunohistochemistry investigations in the liver and spleen established an association between aHEV and the observed lesions in the affected birds, consolidating the knowledge on the pathogenesis of aHEV, which is still largely unknown. Thus, the present investigation extends the natural host range and genotypes of aHEV and strengthens knowledge on the pathogenesis of HSS.

A genetically novel avian Hepatitis E virus in China.

Hepatitis E virus (HEV) infection has a global distribution with diverse hosts, including mammals and avians. In this study, an avian Hepatitis E virus (aHEV) strain with a high mortality rate of about 30%, designated as SDXT20, was obtained from the liver of 30-week-old Hubbard chickens with severe hepatosplenomegaly in 2020 in Eastern China and HEV was proved to be the only pathogen by next-generation sequencing. Its complete genome, which encodes three open reading frames (ORFs), is 6649 nt in length. ORF1-3 encodes three proteins with lengths of 1532 aa, 606 aa, and 82 aa, respectively, and ORF2 and ORF3 overlap with each other. BLAST-based similarity analysis of the complete viral genome demonstrated that SDXT20 had merely 80.5-92.2% similarity with avian Avihepevirus magniiecur strains and 50.4%-54.8% lower similarity with Paslahepevirus balayani, Rocahepevirus ratti, and Chirohepevirus eptesici species. Further genetic evolution analysis of the complete genome and ORF2 revealed that the isolate was genetically distinct from known aHEVs, and it belonged to a novel genetically distinct aHEV. This study provides data for further analysis of the multi-host and cross-host genetic evolution of HEVs.

ICTV Virus Taxonomy Profile: Hepeviridae 2022.

The family Hepeviridae includes enterically transmitted small quasi-enveloped or non-enveloped positive-sense single-stranded RNA viruses infecting mammals and birds (subfamily Orthohepevirinae) or fish (Parahepevirinae). Hepatitis E virus (genus Paslahepevirus) is responsible for self-limiting acute hepatitis in humans; the infection may become chronic in immunocompromised individuals and extrahepatic manifestations have been described. Avian hepatitis E virus (genus Avihepevirus) causes hepatitis-splenomegaly syndrome in chickens. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hepeviridae, which is available at www.ictv.global/report/hepeviridae.

Research Note: The prevalence and vertical transmission of avian hepatitis E virus novel genotypes in Tai'an city, China.

To investigate the prevalence of avian hepatitis E virus (HEV) in chickens and gather evidence of viral vertical transmission, we collected 288 cloacal swabs and 288 yolks samples from 12 farms with clinically healthy chickens in 4 different areas in Tai'an City, Shandong Province, China (i.e., Daiyue District, Xintai City, Feicheng City, and Ningyang County). We also collected 240 samples from 2 breeder farms (from each of which 30 chicks, 30 dead embryos, 30 live embryos, and 30 hatching eggs were taken). PCR detection revealed that the positive rates of cloacal swabs and yolks were 6.25% (18/288) and 4.51% (13/288), respectively. Besides, avian HEV was detected with higher positive rates in the chicks (11.67%), hatching eggs (10.00%), live embryos (13.33%), and dead embryos (26.67%) from 2 breeder farms. Sequence and genetic evolution analyses revealed that the nucleotide homology of the isolated strains was 76.4to 83.9% compared with 4 reported genotypes, but the isolated strains were located in a separate branch, indicating they were potential novel genotypes. In conclusion, those results indicate that the latent infection of avian HEV novel genotypes has been widespread in chicken farms in Tai'an City, and provide reliable evidence of the possible vertical transmission of avian HEV.

Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland.

Orthohepevirus B, commonly known as avian hepatitis E virus (aHEV), causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS) in chickens. BLS is an emerging disease among chicken flocks in several countries around the world. In our previous studies, serology and molecular biology screening revealed that chicken flocks are widely affected by aHEV in Poland. The present study, which was conducted between 2019 and 2020, aimed to investigate the prevalence of aHEV in chicken flocks and other poultry, including ducks, geese, and turkeys. A total of 307 flocks were examined. In addition, 29 samples from captive wild birds (western capercaillies, Tetrao urogallus) were analyzed. In all the investigated poultry species, except turkeys, the nucleic acid sequence covering part of the ORF1 gene of the aHEV genome was detected (34/336 samples, 10.1%). The infection rate was found to be the highest in broiler breeder chicken flocks (14/40 samples; 35%). Phylogenetic analysis of partial ORF1 gene, which encodes helicase, revealed that the obtained sequences belonged to genotypes 2 and 4, while one belonged to genotype 3. Genotype 2 was detected for the first time in domestic geese and ducks, and genotype 4 was detected for the first time in Poland. The study demonstrated the presence of aHEV among the investigated western capercaillies, suggesting that this species is susceptible to aHEV infections and biosecurity is therefore required in western capercaillie breeding facilities.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/ß1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/ß1 integrins. The triggering of the signaling pathway-associated Integrin α5/ß1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

Experimental Cross-Species Transmission of Rat Hepatitis E Virus to Rhesus and Cynomolgus Monkeys.

Rat hepatitis E virus (rat HEV) was first identified in wild rats and was classified as the species Orthohepevirus C in the genera Orthohepevirus, which is genetically different from the genotypes HEV-1 to HEV-8, which are classified as the species Orthohepevirus A. Although recent reports suggest that rat HEV transmits to humans and causes hepatitis, the infectivity of rat HEV to non-human primates such as cynomolgus and rhesus monkeys remains controversial. To investigate whether rat HEV infects non-human primates, we inoculated one cynomolgus monkey and five rhesus monkeys with a V-105 strain of rat HEV via an intravenous injection. Although no significant elevation of alanine aminotransferase (ALT) was observed, rat HEV RNA was detected in fecal specimens, and seroconversion was observed in all six monkeys. The partial nucleotide sequences of the rat HEV recovered from the rat HEV-infected monkeys were identical to those of the V-105 strain, indicating that the infection was caused by the rat HEV. The rat HEV recovered from the cynomolgus and rhesus monkeys successfully infected both nude and Sprague-Dawley rats. The entire rat HEV genome recovered from nude rats was identical to that of the V-105 strain, suggesting that the rat HEV replicates in monkeys and infectious viruses were released into the fecal specimens. These results demonstrated that cynomolgus and rhesus monkeys are susceptible to rat HEV, and they indicate the possibility of a zoonotic infection of rat HEV. Cynomolgus and rhesus monkeys might be useful as animal models for vaccine development.

Isolation, identification and genome analysis of an avian hepatitis E virus from white-feathered broilers in China.

Avian hepatitis E virus (HEV) is the major causative pathogen of the big liver and spleen disease, hepatitis-splenomegaly syndrome, and hepatic rupture hemorrhage syndrome. Until now, there are 6 different avian HEV genotypes that infect chickens have been reported worldwide. Epidemiologic investigations of the avian HEV demonstrated that avian HEV has been widely spread in China in recent years. In this study, an avian HEV named YT-aHEV was obtained from white-feathered broilers using LMH cells by virus isolation assay in Shandong province, China. The complete genome consists of 6656-nt excluding the poly(A) tail. The isolate was highly similar to the CaHEV strain and segregated into the same branch belonging to avian HEV genotype 3. Indirect immunofluorescence using capsid protein-specific polyclonal antibodies confirmed that YT-aHEV could establish productive infection and replicate stably in LMH cells. Furthermore, an in vivo avian HEV infection model was established successfully in specific pathogen-free chicken embryos by intravenous experiments. In the present study, we demonstrate an avian HEV infection associated with liver lesions of hemorrhage and swelling by LMH cells for the first time in a white-feather broiler flock in China. This research also provides a new diagnosis method for detection of avian HEV, which laid a foundation for the understanding of pathogenicity and molecular biology of this virus for further study.

Antigenic cross-reactivity among human, swine, rabbit and avian hepatitis E virus capsid proteins.

Hepatitis E virus (HEV), a zoonotic virus, infects many animal species, including humans. Capsid proteins of human, swine, rabbit and avian HEVs share 48 %-100 % amino acid identity. In the present study, antigenic cross-reactivity among human, swine, rabbit and avian HEV capsid proteins were analyzed in detail using indirect and blocking enzyme-linked immunosorbent assays (ELISAs). The C-terminal 268 amino acids of genotype 1 human, genotype 4 swine, genotype 3 rabbit and genotype B3 avian HEV capsid proteins served as coating antigens for ELISA. Hyperimmune rabbit antisera (against four HEV capsid proteins) and human, pig, rabbit and chicken clinical sera were as primary antibodies. Closely correlated and statistically indistinguishable results were obtained for detection of anti-HEV antibodies in human and pig sera using human, swine and rabbit HEV capsid proteins as coating antigens. Moderately correlated differences in detection of anti-HEV antibodies in rabbit sera were obtained using the three capsid proteins. Statistically significant differences with no correlations were obtained for anti-HEV antibodies detection in chicken sera between avian HEV capsid protein and human, swine and rabbit ones. Blocking ELISA results demonstrated that two common epitopes among the four species HEVs were immunodominant in avian HEV, but were non-immunodominant in human, swine and rabbit HEVs. Nevertheless, three epitopes common to human, swine and rabbit HEVs were all immunodominant epitopes for the three species HEVs. Collectively, these results demonstrate that anti-HEV antibodies in human and pig sera can be detected using human, swine and rabbit HEV capsid proteins. By contrast, for optimal detection of anti-HEV antibodies in rabbit and chicken sera, the respective rabbit and avian HEV capsid proteins should be used. These results provide insights to guide future development of serological assays for diagnosing HEV infections in various animal species.

The diagnosis and molecular epidemiology investigation of avian hepatitis E in Shandong province, China.

BACKGROUND: Avian hepatitis E virus (HEV) is the pathogenic agent of big liver and spleen disease (BLS) and of hepatitis-splenomegaly syndrome (HSS) in chickens, which have caused economic losses to the poultry industry in China. In this study, 18 samples of BLS chickens were collected to reveal the molecular epidemiological characteristics of avian HEV in the province of Shandong, China. RESULTS: Gross and microscopic lesions of clinical samples were observed; then, virology detection and genetic analysis of avian HEV were performed. The results showed that there was significant swelling and rupture in the liver and that the spleen was enlarged. Microscopic lesions demonstrated obvious hemorrhage in the liver, with infiltration of heterophilic granulocytes, lymphocytes, and macrophages, as well as the reduction of lymphocytes in the spleen. Eleven of the 18 samples were positive for avian HEV, with a positive rate of 61.11%. More importantly, all avian HEV-positive samples were mixed infections: among these, the mixed infections of avian HEV and chicken infectious anemia virus (CIAV) and avian HEV and fowl adenovirus (FAdV) were the most common. Furthermore, the genetic evolution analysis showed that all avian HEV strains obtained here did not belong to the reported 4 genotypes, thus constituting a potential novel genotype. CONCLUSIONS: These results of this study further enrich the epidemiological data on avian HEV in Shandong, prove the genetic diversity of avian HEV in China, and uncover the complex mixed infections of avian HEV clinical samples.

Characterisation of the whole genome sequence of an avian hepatitis E virus directly from clinical specimens reveals possible recombination events between European and USA strains.

Avian hepatitis E virus (aHEV) is the causative agent of an important disease of broiler breeders and layers. aHEV cannot be readily propagated in cell culture and is characterised primarily by sequencing of amplicons generated through several RT-PCRs that target individual genes. This study aims to uncover the origin of current Australian aHEV isolates based on whole genome sequencing using clinical liver tissues. Complete genome sequences of the two aHEV isolates were assembled using Nanopore and Illumina reads. The two isolates possessed only four single nucleotide polymorphisms to each other. Comparison of the sequences with aHEV genome sequences available in the GenBank showed the highest nucleotide sequence identity of 88% with the prototype USA strain (AY535004), 82% with the European (AM943647) and genotype 1 Australian strains (AM943647). Recombination analysis suggested that aHEV isolates characterised in this study are progeny of a cross between a US and a Hungarian strain. Phylogenetic tree and phylogenetic networks constructed using complete genome and individual coding sequences revealed that Australian aHEV isolates formed a distinct clade closer to the USA strains and classified as genotype 2 whereas genotype 1 Australian strain clustered together with South Korean strains.

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.