Duck hepatitis A virus utilizes PCBP2 to facilitate viral translation and replication.

Duck hepatitis A virus type 1 (DHAV-1) is an important member of the Picornaviridae family that causes highly fatal hepatitis in ducklings. Since picornaviruses have small genomes with limited coding capacity, they must utilize host proteins for viral cap-independent translation and RNA replication. Here, we report the role of duck poly(rC)-binding protein 2 (PCBP2) in regulating the replication and translation of DHAV-1. During DHAV-1 infection, PCBP2 expression was upregulated. A biotinylated RNA pull-down assay revealed that PCBP2 positively regulates DHAV-1 translation through specific interactions with structural domains II and III of the DHAV-1 internal ribosome entry site (IRES). Further studies revealed that PCBP2 promotes DHAV-1 replication via an interaction of its KH1 domain (aa 1-92) with DHAV-1 3Dpol. Thus, our studies demonstrated the specific role of PCBP2 in regulating DHAV-1 translation and replication, revealing a novel mechanism by which host‒virus interactions regulate viral translation and replication. These findings contribute to further understanding of the pathogenesis of picornavirus infections.

HSP70 positively regulates translation by interacting with the IRES and stabilizes the viral structural proteins VP1 and VP3 to facilitate duck hepatitis A virus type 1 replication.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

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.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1.

BACKGROUND: DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay. METHODS: A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 µg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 µg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings. RESULTS: Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest. CONCLUSION: We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

mRNA and miRNA expression profiles reveal the potential roles of RLRs signaling pathway and mitophagy in duck hepatitis A virus type 1 infection.

Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target.

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).

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.

Surface Display of Duck Hepatitis A Virus Type 1 VP1 Protein on Bacillus subtilis Spores Elicits Specific Systemic and Mucosal Immune Responses on Mice.

Duck viral hepatitis, primarily caused by duck hepatitis A virus type 1 (DHAV-1), poses a significant threat to the global duck industry. Bacillus subtilis is commonly utilized as a safe probiotic in the development of mucosal vaccines. In this study, a recombinant strain of B. subtilis, designated as B. subtilis RV, was constructed to display the DHAV-1 capsid protein VP1 on its spore surface using the outer coat protein B as an anchoring agent. The immunogenicity of this recombinant strain was evaluated in a mouse model through mixed feeding immunization. The results indicated that B. subtilis RV could elicit specific systemic and mucosal immune responses in mice, as evidenced by the high levels of serum IgG, intestinal secretory IgA, and potent virus-neutralizing antibodies produced. Furthermore, the recombinant strain significantly upregulated the expression levels of IL-2, IL-6, IL-10, TNF-α, and IFN-γ in the intestinal mucosa. Thus, the recombinant strain maintained the balance of the Th1/Th2 immune response and demonstrated an excellent mucosal immune adjuvant function. In summary, this study suggests that B. subtilis RV can be a novel alternative for effectively controlling DHAV-1 infection as a vaccine-based feed additive.

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.

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.

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.

The lysine at position 151 of the duck hepatitis A virus 1 2C protein is critical for its NTPase activities.

The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg2+ indispensable and affected by other biochemical characteristics such as Mn2+, Ca2+, Zn2+, Na+ and pH. Guanidine hydrochloride (GdnHCl), a potent inhibitor of viral RNA replication, inhibited ATPase activity of the DHAV-1 2C protein in a dose-dependent manner. Finally, we constructed three mutants to identify the key site for the ATPase activity of the DHAV-1 2C protein. These results indicate that lysine at position 151 of the DHAV-1 2C protein is very important for NTPase activity. Here, we demonstrated and partially characterized that the DHAV-1 2C protein has NTPase activity and showed that mutation of the lysine in the conserved Walker A impairs that activity. The results serve to confirm what is readily predicted from previous work on picornavirus 2C proteins. It also provides a basis for further study of the 2C protein and the function of NTPase activity on the viral life cycle.

The protective effect of baicalin on duck hepatitis A virus type 1-induced duck hepatic mitochondria dysfunction by activating nuclear erythroid 2-related factor 2/antioxidant responsive element signaling pathway.

Duck hepatitis A virus type 1 (DHAV-1) is the main pathogen of duck viral hepatitis, but the efficacy of the licensed commercial vaccine needs to be further improved. Therapeutic measures of specific drugs for DHAV-1-infected ducklings need to be urgently developed. Baicalin possesses good antiviral effects. This study aims to investigate the mechanism of baicalin in protecting hepatic mitochondrial function from DHAV-1. The ELISA method was used to detect changes of hepatic and mitochondrial catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), inducible nitric oxide synthase (iNOS), adenosine triphosphate (ATP), and malondialdehyde (MDA) levels in vivo and vitro. Hematoxylin and eosin sections and transmission electron microscopy were used to observe liver pathological changes and mitochondrial structural changes. The changes in mitochondrial membrane potential were detected by JC-1 staining method. Western blot and quantitative real-time PCR were employed to analyze the gene and protein expressions in the nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway in duck embryonic hepatocytes infected with DHAV-1. Results showed the administration of baicalin increased the survival rate of ducklings, and alleviated hepatic damage caused by DHAV-1 by enhancing the antioxidant enzyme activities of the liver and mitochondria, including SOD, GPX, CAT, and reducing lipid peroxidative damage (MDA content) and iNOS activities. The mitochondrial ultrastructure changed and the significant increase of ATP content showed that baicalin maintained the structural integrity and ameliorated mitochondrial dysfunction after DHAV-1 infection. In vitro, DHAV-1 infection led to loss of mitochondrial membrane potential and lipid peroxidation and decreased antioxidative enzyme activities (SOD, GPX) and mitochondrial respiratory chain complex activities (succinate dehydrogenase, cytochrome c oxidase). Baicalin relieved the above changes caused by DHAV-1 and activated the gene and protein expressions of Nrf2, which activated ARE-dependent genes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1), SOD-1, and GPX-1. In addition, baicalin increased the protein expressions of antioxidative enzymes (SOD, GPX). Hence, baicalin protects the liver against oxidative stress in hepatic mitochondria caused by DHAV-1 via activating the Nrf2/ARE signaling pathway.

Establishment of Duplex SYBR Green I-based Real-time PCR Assay for Simultaneous Detection of Duck Hepatitis A Virus-1 and Duck Astrovirus-3.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 101 copies/µl and 3.78 × 101 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

Artículo regular­Establecimiento de un ensayo dúplex de PCR en tiempo real basado en SYBR Green I para la detección simultánea del virus de la hepatitis A del pato-1 y del astrovirus del pato tipo 3. La hepatitis viral del pato (DVH) afecta principalmente a los patitos menores de 1 mes de edad, causa necrosis hepática, agrandamiento y hemorragia, y es altamente letal, lo que pone en grave peligro la industria del pato. La prevalencia del virus de la hepatitis A del pato (DHAV-1) y del astrovirus del pato tipo 3 (DAstV-3) está aumentando y la coinfección es común. Además, las características clínicas similares de las infecciones por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3 así como la alta frecuencia de coinfección dificultan el diagnóstico. En este estudio, para establecer un método para la detección rápida y simultánea por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3, se diseñaron dos pares de iniciadores específicos según sus regiones génicas conservadas. Se estableció con éxito un ensayo cuantitativo de PCR basado en SYBR® Green I que pudo detectar rápida y diferencialmente los dos virus. Además, el ensayo es muy específico y no muestró reacción cruzada con otros virus comunes. El límite de detección del método fue de 7.34 × 101 copias/µl y de 3.78 × 101 copias/µl para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3, respectivamente, lo que indica una alta sensibilidad. Se analizaron un total de 34 muestras clínicas utilizando el método establecido; las tasas positivas para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3 fueron del 14.71% y 8.82%, respectivamente y la de coinfección fue del 2.94% (1/34), que fue mejor que la obtenida con el método de PCR convencional. En resumen, el ensayo cuantitativo de PCR basado en SYBR Green I establecido en este estudio tiene alta especificidad, buena sensibilidad y precisión, alta viabilidad y es rápido. Por lo tanto, puede ser una herramienta poderosa para la detección de coinfecciones con el virus de la hepatitis A del pato y astrovirus del pato tipo 3 y para futuros estudios epidemiológicos.

Integrated miRNA and mRNA Expression Profiles Reveal Differentially Expressed miR-222a as an Antiviral Factor Against Duck Hepatitis A Virus Type 1 Infection.

Duck hepatitis A virus 1 (DHAV-1) is a highly contagious etiological agent that causes acute hepatitis in young ducklings. MicroRNAs (miRNAs) play important regulatory roles in response to pathogens. However, the interplay between DHAV-1 infection and miRNAs remains ambiguous. We characterized and compared miRNA and mRNA expression profiles in duck embryo fibroblasts cells (DEFs) infected with DHAV-1. In total, 36 and 96 differentially expressed (DE) miRNAs, and 4110 and 2595 DE mRNAs, were identified at 12 and 24 h after infection. In particular, 126 and 275 miRNA-mRNA pairs with a negative correlation were chosen to construct an interaction network. Subsequently, we identified the functional annotation of DE mRNAs and target genes of DE miRNAs enriched in diverse Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may be important for virus resistance, cell proliferation, and metabolism. Moreover, upregulated miR-222a could negatively regulate DHAV-1 replication in DEFs and downregulate integrin subunit beta 3 (ITGB3) expression by targeting the 3' untranslated region (3'UTR), indicating that miR-222a may modulate DHAV-1 replication via interaction with ITGB3. In conclusion, the results reveal changes of mRNAs and miRNAs during DHAV-1 infection and suggest miR-222a as an antiviral factor against DHAV-1.

Icariin and its phosphorylated derivatives reduce duck hepatitis A virus serotype 1-induced oxidative stress and inflammatory damage in duck embryonic hepatocytes through mitochondrial regulation.

Duck hepatitis A virus serotype 1 (DHAV-1) causes acute inflammatory injury with a very high mortality rate in ducklings, leading to severe economic losses worldwide, especially in mainland China. There is an urgent need to find new treatments to prevent and control infection with DHAV-1. Not only is there a shortage of commercial anti-DHAV-1 drugs, but there are also gaps in the use and protection rates of existing commercial vaccines. We previously found that icariin (ICA), an extract of Epimedium, can reduce the mortality rate of ducklings after DHAV-1 infection, and the effect of ICA after phosphorylation modification (pICA) is more evident. In this study, we used duck embryo hepatocytes (DEHs) to investigate the mechanism of the alleviation of DHAV-1-induced inflammation and oxidative stress by ICA and pICA, and to further study their effects on hepatocyte mitochondrial function, apoptosis and cell cycle. It was found that ICA and pICA can inhibit the negative effects of DHAV-1 on apoptosis and cell cycle progression by stabilizing mitochondrial function, thereby reducing inflammation and ultimately protecting liver cells. The effects of pICA are more beneficial than those of ICA. The results of this study may be useful in the development of a new prophylactic and therapeutic strategy against DHAV-1 and other acute inflammatory diseases.

Comparative Pathogenicity of Duck Hepatitis A Virus-1 Isolates in Experimentally Infected Pekin and Muscovy Ducklings.

Duck hepatitis virus (DHV) has always been considered one of the threats endangering duck farming in Egypt since the 1960s. In the current study, suspected DHV samples (n = 30) were obtained from commercial Pekin, Mulard (hybrid), and Muscovy duck farms and backyards in Beheira, Alexandria, Gharbia, Kafr El-Sheikh, and Giza provinces between 2012 and 2017. Diseased 3-21-day-old ducklings showed a clinical history of high mortality rates and nervous signs. Samples were screened by RT-PCR targeting the 5'UTR region and VP1 gene. The PCR-confirmed samples (n = 7) were isolated via allantoic route inoculation onto 9-day-old specific-pathogen-free embryonated chicken eggs. Embryos showed stunting, subcutaneous hemorrhages, and liver necrotic greenish-yellow foci. Duck hepatitis A virus-1 (DHAV-1) isolates were genetically analyzed in comparison to other field and vaccine strains. Phylogenetic analyses of the full-length VP1 gene sequences revealed that the obtained DHAV-1 field isolates clustered into genetic group 4 alongside other Egyptian strains isolated during the same period (95.9-99.72% similarity). Amino acid substitutions in the carboxyl-terminal of VP1 (I180T, G184E, D193N, and M213I) were identified in two strains. Also, deletion mutation at I189 was detected in three DHAV-1 strains. Additionally, the two amino acid residues E205 and N235 were common among the isolated strains and other virulent DHAV-1 strains. Two DHAV-1 isolates originated from Pekin source were selected for conducting the comparative pathogenicity testing based on detected point mutations at C-terminus of VP1. We evaluated the pathogenicity of these isolates by investigating clinical signs, mortality rates, and gross pathological and microscopic lesions. The study revealed that experimentally infected Pekin and Muscovy ducklings showed similar clinical signs including squatting down, lateral recumbency, and spasmodic kicking. Muscovy showed milder pathological changes in the liver compared to Pekin ducklings. Histopathological findings supported the gross pathological lesions detected in both breeds. In conclusion, these data provide updated information on the genetic diversity and pathotyping of Egyptian DHAV-1 strains. To the best of our knowledge, this is the first report of comparative pathogenicity of recent DHAV-1 strains in Pekin and Muscovy ducklings in Egypt and the Middle East region.