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.

Pathogenicity of field strain of fowl aviadenovirus serotype 11 isolated from chickens with inclusion body hepatitis in Morocco.

Outbreaks of inclusion body hepatitis have emerged in Morocco since 2013 and has resulted in significant economic losses to poultry farms. Three isolates of the causative virus, Fowl adenonovirus (FAdV)were characterized from chickens with IBH, but their pathogenicity has never been investigated. In this work, the pathogenicity of an isolate FAdV 11 (MOR300315 strain) was evaluated by inoculating a group of 40 SPF chickens at 3 days of age by oral route. A group of 40 chicks injected with phosphate-buffered saline solution was used as a control group. The infected chickens showed decreased weight gain from 3dpi. Necropsy displayed pallor and enlargement in liver, swelling and slight hemorrhage in kidney and spleen at 6 dpi. Histopathological changes were mainly characterized by severe and extensive hepatic necrosis associated with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The FAdV was reisolated in chicken embryo fibroblast cell culture from liver tissue homogenate of infected chicken from 3 to 6 dpi. Viral DNA was detected by PCR in liver, kidney, spleen and cloacal swabs from 3 to 13 dpi. Antibody response against inoculated FAdV was appeared from 9 dpi. These results confirmed that the FAdV 11 strain is pathogenic in chicken. This study is the first experimental infection of FAdV 11 in chicken in Morocco, which increase our understanding of its pathogenicity in chickens and indicate that preventive measures against FAdV infection in poultry farms should be implemented in Morocco.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

First Clinical Case of Equine Parvovirus-Hepatitis-Related Theiler's Disease in Asia.

Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler's disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (PCR) and in situ hybridization (ISH) assays. The serum, nasal swab, oral swab, and liver biopsy were positive for EqPV-H according to the PCR assay. Genetic analysis of the partial NS1 gene of EqPV-H showed a unique nucleotide substitution, distinct from that in previously deposited strains. EqPV-H DNA was found not only in hepatocytes but also in bile duct epithelium and Kupffer cells, particularly via ISH. To the best of our knowledge, this is the first case of EqPV-H-associated TD in Asia, providing the first clinical evidence for viral shedding from the mouth and nose, and identification of EqPV-H in the liver. This study contributes to a better understanding of the pathological features of EqPV-H-associated TD.

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.

Differential metabolism-associated gene expression of duck pancreatic cells in response to two strains of duck hepatitis A virus type 1.

Several outbreaks of duck hepatitis A virus type 1 (DHAV-1), which were characterized by yellow coloration and hemorrhage in pancreatic tissues, have occurred in China. The causative agent is called pancreatitis-associated DHAV-1. The mechanisms involved in pancreatitis-associated DHAV-1 infection are still unclear. Transcriptome analysis of duck pancreas infected with classical-type DHAV-1 and pancreatitis-associated DHAV-1 was carried out. Deep sequencing with Illumina-Solexa resulted in a total of 53.9 Gb of clean data from the cDNA library of the pancreas, and a total of 29,597 unigenes with an average length of 993.43 bp were generated by de novo sequence assembly. The expression levels of D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in glycine, serine, and threonine metabolism pathways, were significantly downregulated in ducks infected with pancreatitis-associated DHAV-1 compared with those infected with classical-type DHAV-1. These findings provide information regarding differences in expression levels of metabolism-associated genes between ducks infected with pancreatitis-associated DHAV-1 and those infected with classical-type DHAV-1, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1-infection.

Genomic Epidemiology and Evolution of Duck Hepatitis A Virus.

Duck hepatitis A virus (DHAV), an avian picornavirus, causes high-mortality acute disease in ducklings. Among the three serotypes, DHAV-1 is globally distributed, whereas DHAV-2 and DHAV-3 serotypes are chiefly restricted to Southeast Asia. In this study, we analyzed the genomic evolution of DHAV-1 strains using extant GenBank records and genomic sequences of 10 DHAV-1 strains originating from a large disease outbreak in 2004-2005, in Hungary. Recombination analysis revealed intragenotype recombination within DHAV-1 as well as intergenotype recombination events involving DHAV-1 and DHAV-3 strains. The intergenotype recombination occurred in the VP0 region. Diversifying selection seems to act at sites of certain genomic regions. Calculations estimated slightly lower rates of evolution of DHAV-1 (mean rates for individual protein coding regions, 5.6286 × 10-4 to 1.1147 × 10-3 substitutions per site per year) compared to other picornaviruses. The observed evolutionary mechanisms indicate that whole-genome-based analysis of DHAV strains is needed to better understand the emergence of novel strains and their geographical dispersal.

Dual Circulation of Duck Hepatitis A Virus Genotypes 1 and 3 in Egypt.

Duck hepatitis A virus (DHAV) causes acute hepatitis and mortality, resulting in high economic losses in the duck farm industry. The current study describes the outbreak of DHAV in vaccinated duck farms in North Egypt during 2019 and molecular characterization of the 3' untranslated region (UTR) and viral protein VP1 genes. The 30 samples were collected from 7- to 28-day-old commercial Pekin ducks that showed a history of nervous signs and sudden deaths and were on farms in 6 governorates. DHAV was typed by reverse transcription-polymerase chain reaction (RT-PCR) for 3' UTR and VP1 genes and revealed 20 positive farms, with the first detection of DHAV genotype 3 (DHAV-3) in 18 samples and the classic DHAV-1 in 2 samples. The phylogenetic analysis of VP1 and 3' UTR genes of the nine selected strains representative of six governorates revealed that seven strains were clustered with DHAV-3 Chinese and Korean-Vietnamese strains within different subgroups with 92.4%-93.7% amino acid identity; such strains were distinguishable from the vaccine strain of DHAV-1 used in Egypt with 74.4% amino acid identity. The other strains were closely related to the DHAV-1 Asian strain and the vaccine strain used in Egypt with 98.7%-99.6% amino acid identity for the VP1 gene with different clustering than that of recently isolated DHAV-1 Egyptian strains. The VP1 gene of DHAV-3 had 1 hypervariable region (HVR) with 10 amino acid mutations compared with DHAV3/DN2/Vietnam/2011, but DHAV-1 had 3 HVRs with 1 amino acid mutation in HVRII compared with the DHAV-1 vaccine strain. In conclusion, a new introduction of DHAV-3 with the classical DHAV-1 was recorded in Pekin duck farms in North Egypt that is genetically distant from the vaccinal strain.

Artículo regular­Circulacíon dual de los genotipos 1 y 3 del virus de la hepatitis A del pato en Egipto. El virus de la hepatitis A del pato (con las siglas en inglés DHAV) causa hepatitis aguda y mortalidad, lo que genera grandes pérdidas económicas en la industria de la críanza de patos. El estudio actual describe un brote del virus de la hepatitis A del pato en una granja de patos vacunados en el norte de Egipto durante el año 2019 y la caracterización molecular de los genes de la región no traducida 3' (3' UTR) y la proteína viral VP1. Las 30 muestras se recolectaron de patos Pekin comerciales de 7 a 28 días de edad que presentaban antecedentes de signos nerviosos y muerte súbita y se encontraban en granjas de seis gobernaciones. El virus de la hepatitis A del pato se tipificó mediante la transcripción inversa y reacción en cadena de la polimerasa (RT-PCR) para los genes 3' UTR y VP1 y reveló 20 granjas positivas, con la primera detección del genotipo 3 del virus de la hepatitis A del pato (DHAV-3) en 18 muestras y la detección del virus clásico de la hepatitis A del pato tipo1 en dos muestras. El análisis filogenético de los genes VP1 y 3' UTR de las nueve cepas seleccionadas representativas de seis provincias reveló que siete cepas se agruparon con cepas del virus de la hepatitis A del pato 3 chinas y coreano-vietnamitas dentro de diferentes subgrupos con una identidad de aminoácidos del 92.4% al 93.7%; dichas cepas se distinguían de la cepa vacunal del virus de la hepatitis A del pato tipo 1 utilizada en Egipto con 74.4% de identidad de aminoácidos. Las otras cepas estaban estrechamente relacionadas con la cepa asiática del virus de la hepatitis A del pato tipo 1 y la cepa de vacuna utilizada en Egipto con 98.7% -99.6% de identidad de aminoácidos para el gene VP1 con agrupaciones diferentes a las de las cepas egipcias de virus de la hepatitis A del pato tipo 1 aisladas recientemente. El gene VP1 del virus de la hepatitis A del pato tipo 3 tenía una región hipervariable (HVR) con 10 mutaciones en la secuencia de aminoácidos en comparación con la cepa DHAV3/ DN2/Vietnam/2011, pero el virus de la hepatitis A del pato tipo 1 tenía tres regiones hipervariables con una mutación de aminoácidos en la zona hipervariable II en comparación con la cepa de vacuna virus de la hepatitis A del pato tipo 1. En conclusión, se registró una nueva introducción del virus de la hepatitis A del pato tipo 3 con el virus de la hepatitis A del pato clásico tipo 1 en granjas de patos Pekín en el norte de Egipto, que está genéticamente distante de la cepa vacunal.

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.

Circovirus in Blood of a Febrile Horse with Hepatitis.

Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse's serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73-74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62-63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1's genome sequence.

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.

Serological evidence of avian HEV antibodies in apparently healthy chickens in southwest Nigeria.

Avian hepatitis E virus (aHEV) is associated with hepatitis-splenomegaly syndrome, big liver and spleen disease and hepatic rupture haemorrhage syndrome. However, the knowledge about aHEV in commercial layer chickens in Nigeria is scarce. In this study, 460 serum samples obtained from 36 apparently healthy commercial layer chicken flocks in three states (Ogun, Osun and Oyo States) of southwestern Nigeria were analysed by enzyme linked immunosorbent assay for the presence of anti-aHEV immunoglobulin Y (IgY) antibodies. In total, the overall seroprevalence of anti-aHEV antibodies was 14.6%. The serological analysis revealed that 75% of the flocks examined were positive for anti-aHEV IgY antibodies from chickens of various ages in all three states. The percentage of the seropositive chickens in the three states varied from flock to flock ranging from 60% to 88.8% and seropositive chickens were detected at any age (24-52 weeks of age) without significant differences between the age groups. This is the first report assessing the presence of aHEV antibodies in chickens from Nigeria. The detection of anti-aHEV antibodies in commercial layer chickens in this study emphasizes the importance of serosurveillance in disease monitoring due to the economic threat posed by aHEV as a result of decreased egg production and increased mortality in affected commercial layer chicken farms. However, further studies are essential to reveal the clinical implications and to assess the real burden of aHEV in Nigeria.

Evidence of possible vertical transmission of duck hepatitis A virus type 1 in ducks.

Duck hepatitis A virus (DHAV) causes a highly contagious and acute disease in ducklings younger than 3 weeks of age and spreads rapidly by horizontal transmission to all susceptible ducklings in the flock. To date, there is no evidence of vertical transmission of DHAV-1. In a previous study, we identified a novel DHAV type 1 (DHAV-1) isolate that could infect adult ducks and induce laying drop. In this study, 30 non-embryonated duck eggs and 60 17-day-old embryos were collected from three breeding duck flocks with egg drop syndrome caused by DHAV-1 in China, and 30 17-day-old embryos were randomly selected from the 60 embryos and allowed to hatch. DHAV-1 RNA was detected by RT-PCR in 10 of 30 non-embryonated eggs, 9 of 30 17-day-old embryos, 5 of 7 dead embryos and 5 of 23 newly hatched ducklings. Overall, 29 of 90 (32.2%) eggs and embryos were positive for DHAV-1. Three DHAV-1 strains were isolated from the dead duck embryos of the three breeding duck flocks, respectively. Pathogenicity studies showed that the three DHAV-1 isolates had median embryo lethal doses but were highly pathogenic to healthy ducklings. Compared with the DHAV reference strains, there were two specific amino acid mutation sites (F169 and S220 ) in VP1 of the three isolates. To the best of our knowledge, this is the first report that DHAV-1 is isolated from duck embryos. The findings provide evidence of possible vertical transmission of DHAV-1 from breeding ducks to ducklings.

Research Note: A putative novel subtype of the avian hepatitis E virus of genotype 3, Jiangxi province, China.

In recent years, the avian hepatitis E virus (HEV) has been widely spread in China, causing huge economic losses. Several studies have carried out detailed epidemiologic investigations of the avian HEV, but no data were from Jiangxi province. Since early April 2020, diseases similar to hepatic rupture hemorrhage syndrome caused by the avian HEV occurred in a Roman Brown layer farm in Jiangxi province, indicating this virus may also be epidemic there. To make this assumption clear, 20 liver samples were collected from the sick flock and then analyzed by detailed viral detection, which confirmed that the avian HEV should be responsible for the aforementioned disease (6 of 20). Then, the capsid gene of the virus was sequenced to show the molecular characteristics of the strain circulating in the aforementioned flock. Sequence comparison showed that it shared 80.7 to 94.7% identities with 12 published strains, while phylogenetic analysis confirmed that it belongs to a new subtype of genotype 3. Moreover, basing on a 242 bp fragment, the novel also shared high similarities to reference strains identified as genotypes before, revealing the genotype 3 maybe very popular in China and even can be divided into several subgroups. In conclusion, a novel avian HEV strain was identified in this study, which belongs to a new subtype of genotype 3. The analysis makes up for the molecular epidemiologic data of avian HEV and provides a basis for further understanding the spread of avian HEV in China.

First Report of Duck Hepatitis A Virus 3 from Duckling Flocks of Egypt.

Duck hepatitis A viruses (DHAV-1, DHAV-2, and DHAV-3) are the predominant causes of duck virus hepatitis (DVH), a disease of ducklings that leads to massive morbidities, mortalities, and economic losses. As a duck-producing country, Egypt suffered lately from several attacks of DVH, despite the regular vaccination of birds. Between Spring 2016 and Summer 2018, 54 duckling flocks in the Sharkia province of Egypt were tested using the reverse-transcription PCR (RT-PCR) based on the DHAV-3D targeting primers. Of them, 27.8% (15/54) were positive. Upon retesting of positive samples using RT-PCR and duck hepatitis A virus (DHAV)-3 VP1-based primers, 33.3% (5/15) contained DHAV-3 RNA. For further analysis at the molecular level, the VP1 and the 3D genes were sequenced using the same primer sets used earlier. The phylogenetic trees confirmed that study sequences belonged to DHAV-3. However, they were displayed as a separate cluster following a geographically dependent distribution. They were also completely unrelated to the Egyptian DHAV-1-based vaccine. This was further confirmed by low nucleotide and amino acid identities in relation to this vaccine. In addition, the VP1 and 3D genes had the same phylogenetic topography. The study VP1 sequences had three unique amino acid substitutions (L59, V208 only in one strain, and C219). As far as we know, this is the first report on DHAV-3 outside Asia, particularly in Egypt. Accordingly, the vaccination strategy against DHAV should be quickly updated to avoid further dissemination of the virus. The epidemiology, pathogenicity, and evolution of DHAV-3 should be carefully monitored in Egypt.

Fowl Adenoviruses Type 8b Isolated from Chickens with Inclusion Body Hepatitis in Japan.

Fowl adenovirus (FAdV) type 8b isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2018 to 2019 were characterized serologically and genetically. Serologically, all isolates were well neutralized by antisera against the FAdV-8b strain, but they were not neutralized by antisera against the FAdV-8a strain. Phylogenetic analysis of the part of the hexon protein gene that includes the L1 region revealed that these isolates were all identical. They were also identical to foreign strains such as the SD1356 strain isolated in China and belonged to FAdV-8b. Furthermore, the 2018-19 Japanese IBH 8b isolates were genetically identical to the SD1356 strain by phylogenetic analysis of fiber genes, but they were different from previous Japanese 8b strains. These findings suggest that the 2018-19 Japanese IBH isolates might have been introduced from other countries.

Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus).

Hepatitis E virus (HEV) is a public health concern because of its zoonotic potential; however, the host species spectrum and the genetic diversity of HEV in many birds are unknown. In the present study, a novel genotype avian HEV was isolated from a bird, silkie fowl, and designated CHN-GS-aHEV (GenBank No. MN562265). The genome of CHN-GS-aHEV was analyzed in comparison with other avian HEVs' and the pathogenicity in silkie fowl was characterized. The results show that the CHN-GS-aHEV shares about 81 % identity with known avian HEV in chickens, ORF3 shares the highest identity (85.1 %-88.0 %) at the nucleotide level, while ORF2 shares the highest identity (96.5 %-98.0 %) at the amino acid level, indicating that the CHN-GS-aHEV belongs to a new genotype avian HEV. The pathogenicity study showed that silkie fowl experimentally infected with the CHN-GS-aHEV demonstrated seroconversion, viremia, fecal virus shedding, liver lesions, and increased ALT level. Furthermore, ultrastructural changes in hepatocyte cells by transmission electron microscopy were characterized by the loss of mitochondrial cristae and swollen mitochondria and endoplasmic reticulum in the infected birds, suggesting that these two organelles may play a significant role in HEV replication. Overall, this study reports the complete genome characterization of a novel avian HEV and successful experimental infection in silkie fowl, and may be serving as a prominent indicator for additional avian HEV detection in other species.