RESUMO
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.
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Vírus da Hepatite do Pato , Hepatite Viral Animal , Infecções por Picornaviridae , Doenças das Aves Domésticas , Animais , Interleucina-2/genética , RNA Polimerase Dependente de RNA/genética , Regulação da Expressão Gênica , RNA Viral/genética , Patos/genética , Infecções por Picornaviridae/veterináriaRESUMO
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.
Assuntos
Vírus da Hepatite do Pato , Hepatite Viral Animal , Animais , Pontos de Checagem do Ciclo Celular , Patos , Vírus da Hepatite do Pato/genética , Fase SRESUMO
Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.
Assuntos
Vírus da Hepatite do Pato , Hepatite Viral Animal , Infecções por Picornaviridae , Doenças das Aves Domésticas , Animais , Autofagia , Proteína Beclina-1 , Patos , Vírus da Hepatite do Pato/fisiologia , Infecções por Picornaviridae/veterináriaRESUMO
Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).
Assuntos
Patos , Vírus da Hepatite do Pato/patogenicidade , Hepatite Viral Animal/virologia , Pancreatite Necrosante Aguda/veterinária , Infecções por Picornaviridae/veterinária , Doenças das Aves Domésticas/virologia , Animais , Vírus da Hepatite do Pato/classificação , Hepatite Viral Animal/patologia , Fígado/patologia , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Pancreatite Necrosante Aguda/virologia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Doenças das Aves Domésticas/patologiaRESUMO
BACKGROUND: Duck viral hepatitis (DVH) is an acute disease of young ducklings with no effective veterinary drugs for treatment. Gynostemma pentaphyllum is a well-known traditional Chinese medicine that plays an important role in the treatment of various diseases. Gypenoside (GP), one of the main ingredients of Gynostemma pentaphyllum, was reported with good hepatoprotective effects. However, its low solubility limits its application in the clinics. To improve its solubility and bioactivity, a phosphorylated derivative of gypenoside (pGP) was prepared by the sodium trimetaphosphate-sodium tripolyphosphate (STMP-STPP) method. An infrared spectroscopy method was applied to analyse the structures of GP and pGP. Then, a methyl thiazolyl tetrazolium (MTT) colorimetric assay was applied to study the hepatocyte protective efficacy of these two drugs against duck hepatitis A virus type 1 (DHAV-1) infection, and qPCR, TUNEL labelling and flow cytometry methods were used to study the relevant hepatocyte protective in vitro. RESULTS: The infrared spectroscopy detection results showed that the phosphorylation modification of GP was successful. The MTT colorimetric assay results showed that both GP and pGP possessed good hepatocyte protective efficacy in vitro, and pGP performed better than GP when the drug was added before or after virus inoculation. Furthermore, the qPCR results revealed that both drugs could effectively inhibit the adsorption (when adding GP and pGP pre-virus inoculation), replication and release of DHAV-1, and the viral inhibition rate of pGP was greater than that of GP. The subsequent TUNEL labelling and flow cytometry assays showed that both GP and pGP could significantly inhibit duck embryo hepatocyte apoptosis induced by DHAV-1, and the inhibition effect of pGP was much stronger than that of GP. CONCLUSIONS: GP exerts good hepatocyte protective efficacy not only by inhibiting the proliferation of DHAV-1 but also by inhibiting duck embryonic hepatocyte apoptosis induced by DHAV-1, and phosphorylation modification significantly improves the antiviral and the anti-apoptotic effects of GP. Therefore, pGP has the potential to be developed into a novel drug against DHAV-1 infection.
Assuntos
Vírus da Hepatite do Pato/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Patos , Gynostemma/química , Hepatite Viral Animal/tratamento farmacológico , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fosforilação , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.
Assuntos
Autofagia/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Vírus da Hepatite do Pato/patogenicidade , Infecções por Picornaviridae/metabolismo , Proteômica/métodos , Animais , Interações Hospedeiro-Patógeno , HumanosRESUMO
CONTEXT: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and lethal disease of young ducklings. However, there is still no effective drug to treat DVH. OBJECTIVE: This study assessed the curative effect on DVH of a flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) as well as the hepatoprotective and antioxidative effects of BLIN. MATERIALS AND METHODS: MTT method was used to test the anti-DHAV-1 ability of BLIN in vitro. We then treated ducklings by BLIN (3 mg per duckling, once a day for 5 days) to evaluate the in vivo efficacy. To study the hepatoprotective and antioxidative roles of BLIN in its curative effect on DVH, we investigated the hepatic injury evaluation biomarkers and the oxidative stress evaluation indices of the ducklings. RESULTS: On duck embryonic hepatocytes, DHAV-1 inhibitory rate of BLIN at 20 µg/mL was 69.3%. The survival rate of ducklings treated by BLIN was about 35.5%, which was significantly higher than that of virus control (0.0%). After the treatment of BLIN, both the hepatic injury and the oxidative stress of infected ducklings alleviated. At the same time, a significant positive correlation (p < 0.05) existed between the hepatic injury indices and the oxidative stress indices. CONCLUSIONS: BLIN showed a significant curative effect on DVH. The antioxidative and hepatoprotective effects of BLIN made great contributions to the treatment of DVH. Furthermore, BLIN is expected to be exploited as a new drug for the clinical treatment of DVH.
Assuntos
Antioxidantes/farmacologia , Antivirais/farmacologia , Patos , Flavonoides/farmacologia , Vírus da Hepatite do Pato/efeitos dos fármacos , Hepatite Animal/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Células Cultivadas , Combinação de Medicamentos , Ginsenosídeos/farmacologia , Glicosídeos/farmacologia , Vírus da Hepatite do Pato/patogenicidade , Hepatite Animal/metabolismo , Hepatite Animal/patologia , Hepatite Animal/virologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Fatores de TempoRESUMO
CONTEXT: The flavonoid prescription baicalin-linarin-icariin-notoginsenoside R1 (BLIN) has a curative effect on duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1). However, the mechanism of this curative effect is not understood. OBJECTIVE: This study investigates the mechanism of the curative effect of BLIN on DVH caused by DHAV-1. We analyzed the anti-DHAV-1 reproduction mechanism and immuno-regulatory effect of BLIN. MATERIALS AND METHODS: The anti-DHAV-1 reproduction effects of BLIN at 20, 10, 5 and 2.5 µg/mL in vitro, as well as the influence of BLIN at 20 µg/mL on DHAV-1 adsorption, replication and release were tested using the qRT-PCR method. The promotion abilities of BLIN at 20, 10, 5 and 2.5 µg/mL on T- and B-lymphocyte proliferation were investigated by the MTT method. IL-2 and IFN-γ levels and total anti-DHAV-1 antibody secretion after treatment with DHAV-1 for 4, 8 and 54 h were determined by ELISA. RESULTS: BLIN showed a dose-dependent DHAV-1 reproduction inhibitory effect. The inhibitory effect was highest at 20 µg/mL, where DHAV-1 adsorption and release were significantly lower. Meanwhile, BLIN at 5 µg/mL significantly increased T and B lymphocyte proliferation. BLIN stimulated total anti-DHAV-1 antibody secretion in ducklings at the dosage of 4 mg per duckling, but did not stimulate IL-2 and IFN-γ secretion significantly. CONCLUSIONS: BLIN inhibits DHAV-1 reproduction by suppressing its adsorption and release. Additionally, BLIN promoted the duckling antiviral response.
Assuntos
Antivirais/farmacologia , Flavonoides/farmacologia , Ginsenosídeos/farmacologia , Glicosídeos/farmacologia , Vírus da Hepatite do Pato/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Patos , Vírus da Hepatite do Pato/crescimento & desenvolvimento , Vírus da Hepatite do Pato/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
To investigate the role of apoptosis in duck viral hepatitis pathogenesis, 4- and 21-d-old ducks were inoculated with duck hepatitis A virus serotype 1 and killed at 2, 6, 12, 24, and 48 h postinfection. TdT-mediated dUTP nick-end labeling was used to detect apoptosis cells. Expression profiles of apoptosis-related genes including caspase-3, -8, -9, and Bcl-2 in spleen, bursa of Fabricius, liver, and the quantity of virus in blood were examined using real-time PCR. The TdT-mediated dUTP nick-end labeling analysis indicated there was a significant difference of apoptotic cells between treatments and controls. The same difference also appeared in virus amount variation in blood during infection. Gene expression analysis revealed that the apoptosis-related gene expression profile was different in the 2 groups, and also different between various organs. This study suggested that apoptosis may play an important role in duck hepatitis A virus serotype 1 infection, and apoptosis suppression might facilitate virus multiplication, resulting in the highest virus concentration in the host.
Assuntos
Apoptose , Patos , Vírus da Hepatite do Pato/fisiologia , Infecções por Picornaviridae/veterinária , Doenças das Aves Domésticas/fisiopatologia , Animais , Bolsa de Fabricius/fisiologia , Bolsa de Fabricius/virologia , Regulação da Expressão Gênica , Vírus da Hepatite do Pato/isolamento & purificação , Marcação In Situ das Extremidades Cortadas/veterinária , Fígado/fisiologia , Fígado/virologia , Especificidade de Órgãos , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/fisiopatologia , Infecções por Picornaviridae/virologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Baço/fisiologia , Baço/virologia , Replicação ViralRESUMO
Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.
Assuntos
Patos , Vírus da Hepatite A/genética , Hepatite A/veterinária , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Proteínas Estruturais Virais/genética , Virologia/métodos , Animais , Hepatite A/virologia , Vírus da Hepatite A/classificação , Vírus da Hepatite A/isolamento & purificação , Vírus da Hepatite A/metabolismo , Dados de Sequência Molecular , Filogenia , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterinária , Proteínas Estruturais Virais/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Vírus da Hepatite B do Pato , Vírus da Hepatite do Pato , Doenças das Aves Domésticas , Animais , Vírus da Hepatite do Pato/genética , Patos , Proteínas do Capsídeo/genética , Adenoviridae/genética , Galinhas , Proteínas Recombinantes/genética , Proteínas ViraisRESUMO
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.
Assuntos
Patos , Vírus da Hepatite do Pato , Hepatite Viral Animal , Infecções por Picornaviridae , Doenças das Aves Domésticas , Animais , Feminino , Camundongos , Gravidez , Cisteína , Patos/genética , Patos/virologia , Fator 88 de Diferenciação Mieloide/genética , Infecções por Picornaviridae/veterinária , Placenta , Transdução de Sinais , Receptor 7 Toll-Like/genética , Células HEK293 , HumanosRESUMO
DNAzymes effectively inhibit the expression of viral genes. Duck hepatitis A virus type-1 (DHAV-1) genomic RNA carries an internal ribosome entry site (IRES). The IRES initiates the translation of DHAV-1 via a mechanism that differs from that of cap-dependent translation. Therefore, it is an attractive target for the treatment of DHAV-1. In this study, we designed 6 DNAzymes (Dzs) specifically targeting 300-618 nt sequence in the DHAV-1 5'untranslated region (UTR; a predicted IRES-like element). In the presence of divalent metal ions, three designed DNAzymes (DZ369, DZ454, and DZ514) efficiently cleaved the 300-618 nt sequence of the DHAV-1 5'UTR RNA. The activity of the Dzs was particularly dependent on Mg2+ ions. Subsequently, the translation inhibitory activity of these Dzs was determined by western blotting experiments. The Dzs effectively inhibited the translation mediated by the 300-618 nt of DHAV-1 5'UTR in duck embryo fibroblasts (DEFs). Importantly, DZ454 showed the strongest inhibitory effect, and its inhibition was time and dose dependent. However, none of the Dzs showed significant inhibition of cap-dependent translation. These results suggest that these Dzs show specificity for target RNA. Moreover, DZ454 inhibited the replication of DHAV-1. In conclusion, the designed DNAzymes can be used as inhibitors of DHAV-1 RNA translation and replication, providing new insights useful for the development of anti-DHAV-1 drugs.
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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.
Assuntos
Patos , Vírus da Hepatite do Pato/fisiologia , Hepatite Viral Animal/transmissão , Transmissão Vertical de Doenças Infecciosas/veterinária , Infecções por Picornaviridae/veterinária , Doenças das Aves Domésticas/transmissão , Sequência de Aminoácidos , Animais , China , Vírus da Hepatite do Pato/genética , Hepatite Viral Animal/virologia , Filogenia , Infecções por Picornaviridae/transmissão , Infecções por Picornaviridae/virologia , Doenças das Aves Domésticas/virologia , Alinhamento de SequênciaRESUMO
With the continuous development of duck farming and the increasing breeding density, the incidence of duck hepatitis A virus type 1 (DHAV-1) has been on the rise, seriously endangering the development of duck farming. To reduce the use of antibiotics in duck breeding, susceptibility risks and mortality, and avoid virulence recovery and immune failure risk, this study aims to develop a new type of mucosal immune probiotics and make full use of molecular biology techniques, on the level of genetic engineering, to modify Lactococcus lactis (L. lactis). In this study, a secretory recombinant L. lactis named MG1363-VP1 with an enhanced Green Fluorescent Protein (eGFP) and translation enhancer T7g10L was constructed, which could express the VP1-eGFP fusion protein of DHAV-1. The animal experiment in ducklings was performed to detect the immune response and protection effect of oral microecologics by recombinant L. lactis. The results showed that oral L. lactis MG1363-VP1 significantly induced the body's humoral immune system and mucosal immune system to produce specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) for DHAV-1 in ducklings, and cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon gamma (IFN-γ). The mortality rate was monitored simultaneously by the natural infestation in the process of production and breeding; notably, the ducklings vaccinated with L. lactis MG1363-VP1 were effectively protected against the nature infection of DHAV-1. The recombinant L. lactis MG1363-VP1 constructed in this study provides a new means of preventing and controlling DHAV-1 infection in the future.
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Duck hepatitis A virus type 1 (DHAV-1) is one of the most deadly pathogens that endanger the duck industry. Most viruses usually turn off host translation after infection to facilitate viral replication and translation. For the first time report to our knowledge, DHAV-1 can induce eIF2α phosphorylation and inhibit cellular translation in duck embryo fibroblasts (DEFs). Moreover, the activity of DHAV-1 in the cells caused obvious eIF2α phosphorylation, which has nothing to do with the viral protein. Subsequently, we screened two kinases (PERK and GCN2) that affect eIF2α phosphorylation through inhibitors and shRNA. Notably, the role of GCN2 in other picornaviruses has not been reported. In addition, when the phosphorylation of eIF2α induced by DHAV-1 is inhibited, the translation efficiency of DEFs restores to a normal level, indicating that DHAV-1 induced cellular translation shutoff is dependent on eIF2α phosphorylation.
RESUMO
To develop an indirect enzyme-linked immunosorbent assay(I-ELISA) method based on 3A protein of duck hepatitis A virus type 1(DHAV-1) for detection of DHAV-1 antibody, the recombinant protein 3A of DHAV-1 was expressed in E.coli and detected by Western blotting with DHAV-1 infected duck serum. A 3A-ELISA method using the expressed 3A protein as coating antigen for the detection of antibodies to DHAV-1 was developed. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:200(6.185 µg/ml), 1:20 and 1:2000, respectively. The optimal blocking buffer was 5% BSA. The cutoff value was determined to be 0.274, and the analytical sensitivity was 1:1280. There was no cross reaction between DHAV-1 infected duck serum and other common pathogenic duck serum, indicating that I-ELISA could be used to detect DHAV-1 infected duck serum. The coefficients of variation(CVs) were lower than 10%. The concordance between the I-ELISA based on the 3A subunit of DHAV-1 and that based on the whole DHAV-1 particle was 92.7%. Taken together, the 3A-ELISA method is a highly sensitive and specific test that could be used for screening for DHAV-1 infection and monitoring DHAV-1 antibody.
Assuntos
Patos/imunologia , Ensaio de Imunoadsorção Enzimática , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite do Pato/imunologia , Hepatite Viral Animal/diagnóstico , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais , Reações Cruzadas , Escherichia coli/genética , Hepatite Viral Animal/imunologia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5' untranslated region (UTR), structured sequence elements within the 3' UTR, and a poly(A) tail at the 3' terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3' UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3' UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.
Assuntos
Vírus da Hepatite do Pato/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Células Cultivadas , Patos , Células HEK293 , Vírus da Hepatite do Pato/genética , Humanos , Proteínas de Ligação a RNA/genética , Replicação Viral/genéticaRESUMO
Duck hepatitis A virus type 1 (DHAV-1) is one of the most common and lethal pathogens in young ducklings. Live-attenuated DHAV vaccine (CH60 strain) developed by passaging in chicken embryos provided effective immune protection for ducklings. However, the accurate mechanism for such adaption in chicken embryos is not fully revealed. Here, we utilize RNA-sequencing to perform global transcriptional analysis of DHAV-1-innoculated embryonated livers along with histopathological and ultrastructural analysis. This study revealed that infection with DHAV-1 strain CH60 is associated with enhanced type I and II interferon responses, activated innate immune responses, elevated levels of suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) accompanied with abnormalities in multiple metabolic pathways. Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage. Our study presents a comprehensive characterization of the transcriptome of chicken embryos infected with DHAV-CH60 and provides insight for in-depth exploration of viral adaption and virus-host interactions.