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.

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.

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.

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.

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.

Duck hepatitis A virus prevalence in mainland China between 2009 and 2021: A systematic review and meta-analysis.

Duck hepatitis A virus (DHAV) is a single-strand positive-sense small RNA virus that causes high mortality in ducklings. In recent years, the incidence of DHAV-3 subtype has been increasing in China, leading to great economic losses to the duck-breeding industry. We investigated the incidence and mortality rates of DHAV in ducks and analysed the seroprevalence of DHAV in mainland China, by meta-analysis. Twenty-six studies published between 2009 and 2021 were retrieved, with a total of 689,549 cases from 14 provinces. Using the DerSimonian-Laird model, DHAV prevalence was estimated with the variance-stabilizing double arcsine transformation. The incidence of DHAV in mainland China was 12 % (95 % confidence interval [CI]: 3-20 %), and the mortality rate was 11 % (95 % CI: 2-19 %), suggesting that the virus was highly virulent and mortality was high. Time analysis showed that DHAV incidence decreased over time. The typing survey showed that strains of DHAV-1 serotype accounted for 38 % (95 % CI: 21-56 %) and strains of DHAV-3 serotype accounted for 49 % (95 % CI: 31-68%) of the tested samples. The decline in the detection rate of DHAV-1 may be due to the widespread use of the DHAV-1 vaccine, which has effectively controlled the DHAV-1 serotype virus. The DHAV-3 vaccine has been on the market for a short time and has no cross protection with DHAV-1, so DHAV-3 accounted for a high proportion of the tested samples. Subgroup analysis of the detection methods showed little difference between PCR and other detection methods.

Duck hepatitis A virus type 1 mediates cell cycle arrest in the S phase.

BACKGROUND: Duck hepatitis A virus type 1 (DHAV-1) is one of the most serious pathogens endangering the duck industry. However, there are few studies on the regulation of the cell cycle by DHAV-1. METHODS: In this study, flow cytometry was applied to analyze the effect of DHAV-1 infection on the cell cycle of duck embryo fibroblasts (DEFs). Subsequently, we analyzed the effects of cell cycle phases on DHAV-1 replication by real-time reverse transcriptase quantitative PCR (real-time RT-qPCR). RESULTS: Flow cytometry data analysis found that DEFs in the S phase increased by 25.85% and 54.21% at 24 h and 48 h after DHAV-1 infection, respectively. The levels of viral RNA detected by real-time RT-qPCR were higher in the DEFs with synchronization in the S phase or G0/G1 phase than in the control group. However, there was no difference in viral copy number between the G2/M phase arrest and control groups. In addition, non-structural protein 3D of DHAV-1 significantly increased cells in the S phase, indicating that 3D protein is one of the reasons for the cell cycle arrest in the S phase. CONCLUSIONS: In summary, DHAV-1 infection induces the cell cycle arrest of DEFs in the S phase. Both S phase and G0/G1 phase synchronization facilitate the replication of DHAV-1, and 3D protein is one of the reasons for the S phase arrest.

Attenuated Duck Hepatitis A Virus Infection Is Associated With High mRNA Maintenance in Duckling Liver via m6A Modification.

Host translation is generally modulated by viral infection, including duck hepatitis A virus (DHAV) infection. Previously, we reported that cellular protein synthesis in a cell model of duck embryo fibroblasts is significantly inhibited by DHAV infection but not viral proteins, suggesting that an important viral-host interaction occurs at the translational level. In this study, we aim to further understand the impact of DHAV virulence on cellular N6-methyladenosine (m6A) modification, which is essential to a wide variety of RNA biological processes, such as mRNA stabilization and translation. Using m6A antibody-based immunoprecipitation, m6A-seq, and LC-MS/MS, we observed that m6A-modified mRNA exists in both virulent and attenuated DHAV-infected duckling livers. Importantly, m6A levels in mRNA were much higher in attenuated DHAV-infected livers compared with virulent DHAV-infected livers, suggesting virulence-dependent regulation of m6A modification. Analysis of modification motifs indicated that GAAGAAG is the most enriched motif. Combined m6A-seq and RNA-seq data analysis indicated a generally positive correlation between m6A and mRNA expression levels in DHAV-infected duckling livers. GO analysis of genes with decreased or increased m6A levels showed that these genes were enriched in various terms, including oxidation-reduction processes and antiviral immune responses. Collectively, our work reveals DHAV virulence-dependent coordination between m6A modification and mRNA expression in duckling livers.

Long Noncoding RNA Expression Rofiles Elucidate the Potential Roles of lncRNA- XR_003496198 in Duck Hepatitis A Virus Type 1 Infection.

Duck hepatitis A virus type 1 (DHAV-1) is a highly lethal virus that severely affects the duck industry worldwide. Long noncoding RNAs (lncRNAs) exert crucial roles in pathogen attacks. Here, we conducted deep transcriptome analysis to investigate the dynamic changes of host lncRNAs profiles in DHAV-1-infected duck embryo fibroblasts. We identified 16,589 lncRNAs in total and characterized their genomic features. Moreover, 772 and 616 differentially expressed lncRNAs (DELs) were screened at 12 and 24 h post-infection. Additionally, we predicted the DELs' cis- and trans-target genes and constructed lncRNA-target genes regulatory networks. Functional annotation analyses indicated that the putative target genes of DELs participated in diverse vital biological processed, including immune responses, cellular metabolism, and autophagy. For example, we confirmed the dysregulation of pattern recognition receptors (TLR3, RIG-I, MDA5, LGP2, cGAS), signal transducers (STAT1), transcription factors (IRF7), immune response mediators (IL6, IL10, TRIM25, TRIM35, TRIM60, IFITM1, IFITM3, IFITM5), and autophagy-related genes (ULK1, ULK2, EIF4EBP2) using RT-qPCR. Finally, we confirmed that one DHAV-1 induced lncRNA-XR_003496198 is likely to inhibit DHAV-1 replication in DEFs. Our study comprehensively analyzed the lncRNA profiles upon DHAV-1 infection and screened the target genes involved in the innate immune response and autophagy signaling pathway, thereby revealing the essential roles of duck lncRNAs and broadening our understanding of host-virus interactions.

Protection of Ducklings from Duck Hepatitis A Virus Infection with ELPylated Duck Interferon-α.

Duck viral hepatitis type I (DVH I) is a lethal disease in ducklings caused by duck hepatitis A virus (DHAV). Although the commercial vaccine is available for vaccination of one-day-old ducklings or breeder ducks, the disease is still prevalent due to the delayed immune response in ducklings and variable maternal antibody levels in breeder duck flocks. To explore the feasibility of duck interferon-α (DuIFN-α) for control of DVH I, DuIFN-α was expressed as an elastin-like polypeptide (ELP) fusion protein (ELP-DuIFN-α) in E. coli and purified by inverse phase transition cycling (ITC). After detection of its cytotoxicity, bioactivity, plasma stability and serum half-life, the protective efficacy of ELP-DuIFN-α against DHAV-1 infection of embryos or ducklings was evaluated using different treatment routes at different infection times. The results show that ELP-DuIFN-α was correctly expressed and purified to more than 90% purity after two cycles of ITC. The purified fusion protein had a specific anti-DHAV-1 activity of 6.0 × 104 IU/mg protein, significantly extended plasma stability and serum half-life without overt cytotoxicity. After allantoic injection with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 5/5, 5/5 or 4/5 embryos survived from the virus challenge. After intramuscular injection or oral administration with ELP-DuIFN-α, 3/5 or 4/5 ducklings survived from co-infection with DHAV-1. After oral administration with ELP-DuIFN-α pre-infection, co-infection or post-infection with DHAV-1, 3/5, 4/5 or 4/5 ducklings survived from the virus challenge, and the relative transcription levels of interferon-stimulated genes were significantly higher than the normal control group and virus challenge control group (p < 0.01). These experimental data suggest that ELP-DuIFN-α can be used as a long-lasting anti-DHAV-1 reagent.

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.

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.

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.

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.

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.

Proteomics reveals the effect of type I interferon on the pathogenicity of duck hepatitis A virus genotype 3 in Pekin ducks.

Duck hepatitis A virus genotypes 3 (DHAV-3) has become the most prevalent pathogen of duck viral hepatitis (DVH) in Asian duck industry in recent years. Previous studies on the pathogenic mechanism of DHAV-3 mainly focused on examine host gene expression levels. However, the study about host protein expression levels has not been reported. For this, proteomics analysis on livers of infected 7-day-old Pekin ducks with DHAV-3 112803 strain was performed to screen differentially expressed proteins. A total of 3,385 proteins were identified, and we found 39 proteins in the challenged group (CH) were significantly up-regulated and 15 proteins were significantly down-regulated in comparison with control group (CON). GO results showed that 9 of the top 20 GO terms were involved in type I interferon regulation, and the KEGG pathway enrichment results showed that innate immune responses were significantly enriched, such as RIG-1-like, Toll-like and NOD-like receptor signaling pathways. Notably, interaction between 11 up-regulation proteins promoted interferon-induced protein synthesis and supported viral genome replication, which could aggravate inflammatory response and liver damage. These findings, together with RT-qPCR verification of related genes, support the view that the type I interferon may play an extremely important role in the pathogenic mechanism of DHAV-3.

Effect of duck hepatitis A virus genotype 3 infection on glucose metabolism of Pekin ducklings and underlying mechanism.

Earlier works identified the second generation (Z8R2) of a resistant Pekin duck line to duck hepatitis A virus genotype 3 (DHAV-3), which displays significantly strong resistance than that of the second generation (Z8S2) of a susceptible Pekin duck line. To understand the genetic mechanisms that determine the different resistance/susceptibility of Z8R2 and Z8S2 to DHAV-3, transcriptome analysis on livers of infected Pekin ducklings was performed to screen differentially expressed genes (DEGs). We found that DHAV-3 infection has a great effect on metabolism of Z8S2 at the transcription level. Using a newly created fourth generation of the resistant Pekin duck line (Z8R4) and an unselected Pekin duck flock (Z7) as models, hypoglycemia and dramatically increased aspartate aminotransferase and alanine aminotransferase were shown to be noticeable signs of fatal cases caused by DHAV-3 infection. These findings, together with expression analysis and verification of DEGs, support the view that DHAV-3 infection results in glucose metabolic abnormalities in susceptible individuals and that there are significant differences in expression patterns of glucose metabolism-related DEGs between susceptible and resistant individuals. Notably, cytokines displayed a negative correlation with glucose synthesis in terms of expression in susceptible individuals following DHAV-3 infection. Mechanism analyses suggests that cytokines will activate PI3K-AKT pathway and/or JAK-STAT pathway by up-regulated expression of JAK2, and thereby causes down-regulated expression of G6PC and/or ACAT1. Cytokines can also cause down-regulated expression of HPGDS by JAK2. The present work contributes to the understanding of pathogenesis of DHAV-3 infection and the resistance breeding project against DHAV-3.

Immune-related miRNA-mRNA regulation network in the livers of DHAV-3-infected ducklings.

BACKGROUND: Duck hepatitis A virus type 3 (DHAV-3) is one of the most harmful pathogens in the duck industry. However, the molecular mechanism underlying DHAV-3 infection in ducklings remains poorly understood. To study the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in DHAV-3 infection in ducklings, we conducted global miRNA and mRNA expression profiling of duckling liver tissues infected with lethal DHAV-3 by high-throughput sequencing. RESULTS: We found 156 differentially expressed miRNAs (DEMs) and 7717 differentially expressed genes (DEGs) in livers of mock-infected and DHAV-3-infected duckling. A total of 19,606 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of these DEMs and DEGs. Moreover, immune-related pathways, including the cytokine-cytokine receptor interaction, apoptosis, Toll-like receptor, Jak-STAT, and RIG-I-like receptor signaling pathway, were significantly enriched through analyzing functions of mRNAs in the network in response to DHAV-3 infection. Furthermore, apl-miR-32-5p, apl-miR-125-5p, apl-miR-128-3p, apl-miR-460-5p, and novel-m0012-3p were identified as potential regulators in the immune-related signaling pathways during DHAV-3 infection. And some host miRNAs were predicted to target the DHAV-3 genome. CONCLUSIONS: This is the first integrated analysis of miRNA and mRNA in DHAV-3-infected ducklings. The results indicated the important roles of miRNAs in regulating immune response genes and revealed the immune related miRNA-mRNA regulation network in the DHAV-3-infected duckling liver. These findings increase our knowledge of the roles of miRNAs and their target genes in DHAV-3 replication and pathogenesis. They also aid in the understanding of host-virus interactions.