Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase.

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3'-untranslated region (3'-UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3'-UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3'-UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Whole-transcriptome sequencing revealed the role of noncoding RNAs in susceptibility and resistance of Pekin ducks to DHAV-3.

As the most prevalent pathogen of duck viral hepatitis (DVH), duck hepatitis A virus genotype 3 (DHAV-3) has caused huge economic losses to the duck industry in China. Herein, we obtained whole-transcriptome sequencing data of susceptible (S) and resistant (R) Pekin duckling samples at 0 h, 12 h, and 24 h after DHAV-3 infection. We found that DHAV-3 infection induces 5,396 differentially expressed genes (DEGs), 85 differentially expressed miRNAs (DEMs), and 727 differentially expressed lncRNAs (DELs) at 24 hpi in S vs. R ducks, those upregulated genes were enriched in inflammation and cell communications pathways and downregulated genes were related to metabolic processes. Upregulated genes showed high connectivity with the miR-33, miR-193, and miR-11591, and downregulated genes were mainly regulated by miR-2954, miR-125, and miR-146b. With the construction of lncRNA-miRNA-mRNA axis, we further identified a few aberrantly expressed lncRNAs (e.g., MSTRG.36194.1, MSTRG.50601.1, MSTRG.34328.7, and MSTRG.29445.1) that regulate expression of hub genes (e.g., THBD, CLIC2, IL8, ACOX2, GPHN, SMLR1, and HAO1) by sponging those highly connected miRNAs. Altogether, our findings defined a dual role of ncRNAs in immune and metabolic regulation during DHAV-3 infection, suggesting potential new targets for treating DHAV-3 infected ducks.

The role of turmeric and black pepper oil nanoemulsion in attenuating cytokine storm triggered by duck hepatitis A virus type I (DHAV-I)-induced infection in ducklings.

The cytokine storm induced by duck hepatitis A virus type 1 (DHAV-1) infection significantly contributes to severe, rapid deaths and economic losses in the duck industry in Egypt. This study aimed to investigate the potential inhibitory effect of a nanoemulsion containing turmeric and black pepper oil on the immune response and pathogenesis of DHAV-1 in ducklings. A total of 105 ducklings from nonvaccinated breeders were divided into 5 experimental groups, each comprising 21 birds. The negative control group (G1) remained noninfected with DHAV-1 and nontreated with nanoemulsion, while the positive control group (G2) was infected with DHAV-1 but not treated with nanoemulsion. The other 2 groups (G3, the supplemented group which was noninfected with DHAV-1), and group 4 (the prophylactic group G4) which was infected with DHAV-1, both received nanoemulsion throughout the experiment. Group 5 (G5, the therapeutic group), on the other hand, which was infected with DHAV-1 received nanoemulsion only from the onset of clinical signs. At 5 days old, the ducklings in the positive control (G2), the prophylactic (G4), and the therapeutic group (G5) were infected with DHAV-1. All the ducklings in the infected groups exhibited depression, anorexia, and opisthotonos, and their livers displayed various degrees of ecchymotic hemorrhage, liver enlargement, and microscopic pathological lesions. Notably, the positive control group (G2) experienced the most severe and pronounced effects compared to the other infected groups treated with the nanoemulsion. Meanwhile, the viral RNA loads were lower in the liver tissues of the infected ducklings treated with the nanoemulsion (G4, and G5) compared to the positive control group G2. Additionally, the nanoemulsion effectively modulated proinflammatory cytokine expression, antioxidant enzymes, liver enzymes, and lipid profile of treated ducklings. In conclusion, the turmeric and black pepper oil nanoemulsion has the potential to be a therapeutic agent for regulating and modulating the immune response, decreasing DHAV-1-induced cytokine storms, and minimizing mortality and economic losses in the duck business. More research is needed to understand how turmeric and black pepper oil nanoemulsion alleviates DHVA-1-induced cytokine storms and lowers duckling mortality.

2A2 protein of DHAV-1 induces duck embryo fibroblasts gasdermin E-mediated pyroptosis.

The duck hepatitis A virus type 1 (DHAV-1) causes rapid death in ducklings by triggering a severe cytokine storm. Pyroptosis is an inflammatory form of programmed cell death that is directly related to an increase in pro-inflammatory cytokine levels. Only a few studies have explored the mechanisms underlying pyroptosis in virus-infected avian cells. In this study, we established an avian infection model in vitro by infecting duck embryo fibroblasts (DEFs) with the virulent DHAV-1 LY0801 strain. DHAV-1 infection induced pyroptosis in the DEFs by activating gasdermin E (GSDME) protein via caspase-3-mediated cleavage. The genes encoding the different structural and non-structural DHAV-1 proteins were cloned into eukaryotic expression plasmids, and the 2A2 protein was identified as the key protein involved in pyroptosis. The HPLC-tandem mass spectrometry (HPLC-MS/MS) and co-immunoprecipitation (Co-IP) analysis established that DHAV-1 2A2 directly interacted with the mitochondrial anti-viral signaling protein (MAVS) both intracellularly and in vitro. Furthermore, we got the results that N-terminal 1-130 aa of 2A2 was involved in the interaction with MAVS and the C-terminal TM domain of MAVS is necessary for the interaction with 2A2 by Co-IP analysis. To our knowledge, this is the first study to reveal that DHAV-1 protein interacts with host proteins to induce pyroptosis. Our findings provide new insights into the molecular pathogenesis of DHAV-1 infection, and a scientific basis for the prevention and treatment of duck viral hepatitis.

Duck hepatitis A virus type 1 infection induces hepatic metabolite and gut microbiota changes in ducklings.

Duck hepatitis A virus type 1 (DHAV-1) can cause severe liver damage in infected ducklings and is a fatal and contagious pathogen that endangers the Chinese duck industry. The objective of this study was to explore the correlation mechanism of liver metabolism-gut microbiota in DHAV-1 infection. Briefly, liquid chromatography-mass spectrometry and 16S rDNA sequencing combined with multivariate statistical analysis were used to evaluate the effects of DHAV-1 infection on liver metabolism, gut microbiota regulation, and other potential mechanisms in ducklings. In DHAV-1-infected ducklings at 72 h postinfection, changes were found in metabolites associated with key metabolic pathways such as lipid metabolism, sugar metabolism, and nucleotide metabolism, which participated in signaling networks and ultimately affecting the function of the liver. The abundance and composition of gut microbiota were also changed, and gut microbiota is significantly involved in lipid metabolism in the liver. The evident correlation between gut microbiota and liver metabolites indicates that DHAV-host gut microbiome interactions play important roles in the development of duck viral hepatitis (DVH).

Construction and immune evaluation of the recombinant duck adenovirus type 3 delivering capsid protein VP1 of the type 1 duck hepatitis virus.

Adenovirus serves as an excellent viral vector and is employed in vector vaccine research. Duck hepatitis A virus type 1 (DHAV1) and duck adenovirus type 3 (DAdV3) cause significant economic losses in the Chinese duck industry. In this study, we found an excellent exogenous gene insertion site in DAdV3 genome of CH-GD-12-2014 strain, within 3 intergenic regions (IGR). Subsequently, we generated a recombinant duck adenovirus named rDAdV3-VP1-188, which exhibits excellent replication characteristics and immunogenicity of DAdV3 and DHAV1. Animal experiments showed that rDAdV3-VP1-188 can provide 100% protection against the DAdV3 and 80% protection against DHAV1. These results showed that rDAdV3-VP1-188 could induce protection against DAdV3 and DHAV1 in ducks, thus indicating the feasibility of DAdV3 as a vector for the development of avian vector vaccines. These insights contribute to the further development of DAdV3 vectors and other adenovirus vectors.

Plasma lipidome reveals susceptibility and resistance of Pekin ducks to DHAV-3.

Duck hepatitis A virus genotype 3 (DHAV-3) is the most popular pathogen of duck viral hepatitis (DVH) and has led to a huge economic threat to the Asian duck industry. In this work, we investigated the differences in the LC-MS/MS-based dynamic lipid profiles between susceptible and resistant Pekin duck lines with DHAV-3 infection. We found that the plasma lipidome of the two duck lines was characterized differently in expression levels of lipids during the infection, such as decreased levels of glycerolipids and increased levels of cholesteryl esters and glycerophospholipids in susceptible ducks compared with resistant ducks. By integrating lipidomics and transcriptomics analysis, we showed that the altered homeostasis of lipids was potentially regulated by a variety of differentially expressed genes including CHPT1, PI4K2A, and OSBP2 between the two duck lines, which could account for liver dysfunction, apoptosis, and illness upon DHAV-3 infection. Using the least absolute shrinkage and selection operator (LASSO) approach, we determined a total of 25 infection-related lipids that were able to distinguish between the infection states of susceptible and resistant ducks. This study provides molecular clues for elucidating the pathogenesis and therapeutic strategies of DHAV-3 infection in ducklings, which has implication for the development of resistance breeding.

A meta-analysis for vaccine protection rate of duck hepatitis a virus in mainland China in 2009-2021.

BACKGROUND: Duck hepatitis A virus (DHAV) is a single-stranded, positive-strand small RNA virus that causes a very high mortality rate in ducklings. The DHAV-3 subtype incidence rate has recently increased in China, causing great economic losses to the waterfowl breeding industry. We analyzed the protection rate of DHAV vaccines used in mainland China from 2009 to 2021 and evaluated the effectiveness of vaccine prevention and control to reduce the economic losses caused by DHAV to the waterfowl breeding industry. We screened five electronic research databases and obtained 14 studies and patents on the protection efficiency of DHAV-1 and DHAV-3 vaccines. RESULTS: Meta-analysis demonstrated that immunized ducklings produced higher antibody levels and had a significantly higher survival rate than non-immunized ducklings [relative risk (RR) = 12, 95% confidence interval (CI) 6-26, P < 0.01]. The age of the ducks and vaccine valence did not affect protection efficiency. Data source analysis of the vaccine protection rate demonstrated that the vaccines conferred immune protection for ducklings in both small-scale experiments and large-scale clinical conditions. The analysis results revealed that although the vaccines conferred protection, the immune protective effect differed between small-scale experimental conditions and large-scale clinical conditions. This might have been due to non-standard vaccination and environmental factors. CONCLUSIONS: Domestic DHAV vaccines can protect ducklings effectively. The subjects immunized (breeding ducks or ducklings) and vaccine valence had no effect on the protective effect. Both small-scale experiments and large-scale clinical conditions conferred immune protection on ducklings, but vaccine immunization under small-scale experimental conditions had slightly better protective effects than large-scale clinical immunization.

Interleukin-22 facilitates the interferon-λ-mediated production of tripartite motif protein 25 to inhibit replication of duck viral hepatitis A virus type 1.

The innate immune system provides a defense against invading pathogens by inducing various interferon (IFN)-stimulated genes (ISGs). We recently reported that tripartite motif protein 25 (TRIM25), an important ISG, was highly upregulated in duck embryo hepatocyte cells (DEFs) after infection with duck viral hepatitis A virus type 1 (DHAV-1). However, the mechanism of upregulation of TRIM25 remains unknown. Here we reported that interleukin-22 (IL-22), whose expression was highly facilitated in DEFs and various organs of 1-day-old ducklings after DHAV-1 infection, highly enhanced the IFN-λ-induced production of TRIM25. The treatment with IL-22 neutralizing antibody or the overexpression of IL-22 highly suppressed or facilitated TRIM25 expression, respectively. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was crucial for the process of IL-22 enhancing IFN-λ-induced TRIM25 production, which was suppressed by WP1066, a novel inhibitor of STAT3 phosphorylation. The overexpression of TRIM25 in DEFs resulted in a high production of IFNs and reduced DHAV-1 replication, whereas the attenuated expression of IFNs and facilitated replication of DHAV-1 were observed in the RNAi group, implying that TRIM25 defended the organism against DHAV-1 propagation by inducing the production of IFNs. In summary, we reported that IL-22 activated the phosphorylation of STAT3 to enhance the IFN-λ-mediated TRIM25 expression and provide a defense against DHAV-1 by inducing IFN production.

Phosphorylated bush sophora root polysaccharides protect the liver in duck viral hepatitis by preserving mitochondrial function.

In order to ascertain the mechanism underlying the therapeutic efficacy of Bush sophora root polysaccharides (BSRPS) and phosphorylated Bush sophora root polysaccharides (pBSRPS) in the treatment of in duck viral hepatitis (DVH), an investigation was conducted to assess the protective impact of BSRPS and pBSRPS against duck hepatitis A virus type 1 (DHAV-1) induced mitochondrial dysfunction both in vivo and vitro. The BSRPS underwent modification through the utilization of the sodium trimetaphosphate - sodium tripolyphosphate method, and was subsequently characterized though Fourier infrared spectroscopy and scanning electron microscopy. Following this, the degree of mitochondrial oxidative damage and dysfunction was described through the use of fluorescence probes and various antioxidative enzyme assay kits. Furthermore, the utilization of transmission electron microscopy facilitated the observation of alterations in the mitochondrial ultrastructure within the liver tissue. Our findings demonstrated that both BSRPS and pBSRPS effectively mitigated mitochondrial oxidative stress and conserved mitochondrial functionality, as evidenced by heightened antioxidant enzyme activity, augmented ATP production, and stabilized mitochondrial membrane potential. Meanwhile, the histological and biochemical examinations revealed that the administration of BSRPS and pBSRPS resulted in a reduction of focal necrosis and infiltration of inflammatory cells, thereby mitigating liver injury. Additionally, both BSRPS and pBSRPS exhibited the ability to maintain liver mitochondrial membrane integrity and enhance the survival rate of ducklings infected with DHAV-1. Notably, pBSRPS demonstrated superior performance in all aspects of mitochondrial function compared to BSRPS. The findings indicated that maintaining mitochondrial homeostasis is a crucial factor in DHAV-1 infections, and the administration of BSRPS and pBSRPS may mitigate mitochondrial dysfunction and safeguard liver function.

Placenta-specific 8 facilitates the infection of duck hepatitis A virus type 1 by inhibiting the TLR7 MyD88-dependent signaling pathway.

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication.

Establishment and application of a PCR assay for the identification of virulent and attenuated duck plague virus DNA in cotton swabs.

Duck plague is an acute, febrile, and septic infectious disease caused by duck plague virus (DPV), which causes serious harm to the duck industry in China. Ducks latently infected with DPV display a clinically healthy state, which is one of the epidemiological characteristics of duck plague. In the present study, to rapidly distinguish vaccine-immunized ducks from wild virus-infected ducks during production, a PCR assay based on the newly identified LORF5 fragment was developed to effectively and accurately identify viral DNA in cotton swab samples and was used to assess artificial infection models and clinical samples. The results showed that the established PCR method had good specificity and that only the virulent and attenuated DNA of duck plague virus was specifically amplified, as the results for the detection of common duck pathogens (duck hepatitis B virus, duck Tembusu virus, duck hepatitis A virus type 1, novel duck reovirus, Riemerella anatipestifer, Pasteurella multocida, and Salmonella) were negative. The amplified fragments of virulent and attenuated strains were 2,454 bp and 525 bp, and their minimum detection amounts were 0.46 pg and 46 pg, respectively. The detection rate of the virulent and attenuated DPV strains in duck oral and cloacal swabs was lower than that of the gold standard PCR method (GB-PCR, which is unable to distinguish virulent and attenuated strains), and cloacal swabs from clinically healthy ducks were more suitable for detection than oral swabs. In conclusion, the PCR assay established in the present study can be used as a simple and effective method for the clinical screening of ducks that are latently infected with virulent strains of DPV and shedding virus, which can provide technical support for the elimination of duck plague from duck farms.

RNA-seq and microRNA association analysis to explore the pathogenic mechanism of DHAV-1 infection with DEHs.

Duck hepatitis A virus 1 (DHAV-1) is one of the main contagious pathogens that causes rapid death of ducklings. To illuminate the potential of DHAV-1-infected underlying mechanisms, we analyzed the mRNA and microRNA (miRNA) expression profiles of duck embryonic hepatocytes (DEHs) in response to DHAV-1. We found 3410 differentially expressed genes (DEGs) and 142 differentially expressed miRNAs (DEMs) at 36 h after DHAV-1 infection. Additionally, DEGs and the target genes of miRNA expression were analyzed and enriched utilizing GO and KEGG, which may be crucial for immune responses, viral resistance, and mitophagy. For instance, the dysregulation of DDX58, DHX58, IRF7, IFIH1, STING1, TRAF3, CALCOCO2, OPTN, PINK1, and MFN2 in DHAV-1-infected DEHs was verified by RT-qPCR. Then, the association analysis of mRNAs and miRNAs was constructed utilizing the protein-protein interaction (PPI) networks, and the expressions of main miRNAs were confirmed, including miR-132c-3p, miR-6542-3p, and novel-mir163. These findings reveal a synthetic characterization of the mRNA and miRNA in DHAV-1-infected DEHs and advance the understanding of molecular mechanism in DHAV-1 infection, which may provide a hint for the interactions of virus and host.

Nonstructural protein 2A2 from Duck hepatitis A virus type 1 inhibits interferon beta production by interaction with mitochondrial antiviral signaling protein and TANK-binding kinase 1.

Type I interferon (IFN-I) is essential for the regulation of host-virus interactions, and viruses have evolved strategies to escape the host immune response. Duck hepatitis A virus type 1 (DHAV-1) causes severe liver necrosis and hemorrhage, neurological symptoms, and high mortality in ducklings. However, how DHAV-1 interacts with the duck innate immune system remains unclear. In this study, DHAV-1-encoded proteins were cloned, and DHAV-1 2A2 was shown to strongly suppress IFN-ß-luciferase activity, triggered by Sendai virus and polyriboinosinic polyribocytidylic acid [poly(I:C)], along with the transcription of IFN-ß and downstream antiviral genes, including OASL, PKR, and TNF-a. In addition, 2A2 interacts with the central adaptor proteins mitochondrial antiviral signaling (MAVS) and TANK-binding kinase 1 (TBK1) by its N-terminal 1-100 amino acids (aa), thus leading to the inhibition of IFN-ß production. Importantly, the deletion of the N-terminal 1-100 aa region of 2A2 abolished inhibition of IFN-I production. Moreover, the transmembrane domain of the MAVS protein and the ubiquitin domain of TBK1 were demonstrated to be required for interaction with DHAV-1 2A2. These findings revealed a novel strategy by which DHAV-1 hijacks cellular immunosurveillance and provided new insights into controlling the disease.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38 nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

DHAV 3CD targets IRF7 and RIG-I proteins to block the type I interferon upstream signaling pathway.

Duck hepatitis A virus type 1 (DHAV-1) is an acute, highly lethal infectious agent that infects ducklings and causes up to 95% mortality in ducklings up to 1 week of age, posing a significant economic threat to the duck farming industry. Previous studies have found that the proteolytic enzyme 3 C encoded by DHAV-1 can inhibit the IRF7 protein from blocking the upstream signaling pathway of the type I interferon to promote viral replication. However, there are still few studies on the mechanism of DHAV-1 in immune evasion. Here, we demonstrate that the DHAV-1 3CD protein can interact with IRF7 protein and reduce IRF7 protein expression without directly affecting IRF7 protein nuclear translocation. Further studies showed that the 3CD protein could reduce the expression of RIG-I protein without affecting its transcription level. Furthermore, we found that the 3CD protein interacted with the N-terminal structural domain of RIG-I protein, interfered with the interaction between RIG-I and MAVS, and degraded RIG-I protein through the proteasomal degradation pathway, thereby inhibiting its mediated antiviral innate immunity to promote DHAV-1 replication. These data suggest a novel immune evasion mechanism of DHAV-1 mediated by the 3CD protein, and the results of this experiment are expected to improve the understanding of the biological functions of the viral precursor protein and provide scientific data to elucidate the mechanism of DHAV-1 infection and pathogenesis.

Duck hepatitis A virus type 1 transmission by exosomes establishes a productive infection in vivo and in vitro.

Duck hepatitis A virus type 1 (DHAV-1) infection causes an acute and highly fatal disease in young ducklings. Exosomes are nano-sized small extracellular vesicles secreted by various cells, which participate in intercellular communication and play a key role in the physiological and pathological processes. However, the role of exosomes in DHAV-1 transmission remains unknown. In this study, through RT-PCR, WB analysis and TEM observation, the complete DHAV-1 genomic RNA, partial viral proteins, and virions were respectively identified in the exosomes derived from DHAV-1-infected duck embryo fibroblasts (DEFs). The productive DHAV-1 infection was transmitted by exosomes in DEFs, duck embryos, and ducklings, and high titers of neutralizing antibodies completely blocked DHAV-1 infection but did not significantly neutralize exosome-mediated DHAV-1 infection. To the best of our knowledge, this is the first report that exosome-mediated DHAV-1 infection was resistant to antibody neutralization in vivo and in vitro, which might be an immune evasion mechanism of DHAV-1.

Current status and future direction of duck hepatitis A virus vaccines.

Duck viral hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a highly fatal and rapidly spreading infectious disease of young ducklings that seriously jeopardizes the duck industry worldwide. DHAV type 1 (DHAV-1) is the main genotype responsible for disease outbreaks since 1945, and the disease situation is complicated by the emergence and dissemination of a novel genotype (DHAV-3) in some countries in Asia and Africa. Live attenuated DHAV vaccines are widely used to induce a considerable degree of protection in ducklings. Breeder ducks are immunized with inactivated or/and live DHAV vaccines to achieve satisfactory levels of passive immunity in progeny. In addition, novel characteristics of virus transmission, pathogenicity and pathogenesis of DHAV were recently characterized, necessitating the development of new vaccines and effective vaccination programmes against DVH. Therefore, a systematic dissection of the profiles, strengths and shortcomings of the available DHAV vaccines is essential. Moreover, to further increase the efficiency of vaccine production and administration, the development of next-generation DHAV vaccines using cutting-edge technologies is also required. In this review, based on a comprehensive summary of the research advances in the epidemiology, pathogenicity, and genomic features of DHAV, we focus on reviewing and analysing the features of the commercial and experimental DHAV vaccines. We also propose perspectives for disease control based on the specific disease situations in different countries. This review provides essential information for vaccine development and disease control of DVH.

Treatment effects of phosphorylated Chrysanthemum indicum polysaccharides on duck viral hepatitis by protecting mitochondrial function from oxidative damage.

To define the underlying mechanism of the beneficial role of Chrysanthemum indicum polysaccharides (CIPS) and phosphorylated Chrysanthemum indicum polysaccharides (pCIPS) in duck viral hepatitis (DVH), we evaluated the protective effects of the CIPS and pCIPS against DVH in terms of antioxidation and mitochondrial function. Fluorescence probes and several assay kits were used to determine the oxidative stress and mitochondrial dysfunction in vitro and vivo. Additionally, transmission electron microscopy was applied to observe the changes of mitochondrial ultrastructure in the liver tissue. Our results indicate that the CIPS and pCIPS significantly enhanced the survival of duck hepatitis A virus type 1 (DHAV-1) infected ducklings. Moreover, the CIPS and pCIPS suppressed oxidative stress and preserved mitochondrial function, such as enhanced antioxidant enzyme activity, increased ATP production, and stabilized mitochondrial membrane potential (MMP). Meanwhile, the results of hematoxylin-eosin (HE) staining and serum biochemical examination demonstrated that treatment with the CIPS and pCIPS could decrease focal necrosis and infiltration of inflammatory cells, which in turn reducing liver injury. Furthermore, the CIPS and pCIPS were able to preserve liver mitochondrial membrane integrity in DHAV-1 challenged ducklings. Notably, the pCIPS was significantly outperformed the CIPS on all measures of liver and mitochondrial function. These results suggested that mitochondrial homeostasis plays an important role in alleviating oxidative damage in the livers, and the hepatocyte protective effects of the CIPS were enhanced after phosphorylation modification.

Comparative transcriptome reveals the effect of IFITM1 on differential resistance to duck hepatitis A virus genotype 3 in Pekin ducks.

BACKGROUND: Duck viral hepatitis (DVH) has a significant economic impact on duck industry, and duck hepatitis A virus genotype 3 (DHAV-3) is the most prevalent pathogen of DVH in Asian duck industry. The detailed study connecting differentially expressed genes (DEGs) and the differential resistance to DHAV-3 have not been accurately described, although a large numbers of DEGs have been identified by transcriptomic studies. RESULTS: Here, a resistant Pekin duck line (Z8R) and a susceptible Pekin duck line (Z8S) as models, high mortality and dramatically increased aspartate aminotransferase (AST), alanine aminotransferase (ALT) and the expression of immune-related genes of Z8S group were shown to be noticeable signs of cases caused by DHAV-3 infection. Compared with the control (Con) group, 1117 down-regulated DEGs and 612 up-regulated DEGs were found in the Z8S group and 37 down-regulated DEGs and 82 up-regulated DEGs were found in the Z8R group. Ultimately, the expression patterns of 10 DEGs were found to be diametrically opposite in Z8R and Z8S group. Functional analysis revealed that IFITM1 was associated with cell growth suppression, which was considered a key candidate gene. Results of flow cytometry showed that the conserved regions of IFITM1 (213-317 bp) could affected the cell cycle of duck embryo fibroblast (DEF) cells after infection with DHAV-3. Transcriptome and western blot analysis suggested that the CCND1, CCNE1 and CDK6 were significantly up-regulated in susceptible ducks by comparing with Con group. CONCLUSIONS: The hepatic injury and pathogenic outcomes caused by DHAV-3 infection were more severe in Z8S group compared to Z8R. Results of transcriptomics analysis and flow cytometry suggested that DHAV-3 infection can induce cell cycle changes that may be associated with IFITM1 expression level. These data will greatly enhance our understanding of the pathogenesis of DHAV-3 infection in ducklings and have implications for development of resistance breeding.